U.S. patent application number 10/591186 was filed with the patent office on 2008-01-03 for pharmaceutical composition for lowering blood sugar level.
This patent application is currently assigned to PHARMAFRONTIER CO., LTD. Invention is credited to Akira Hirasawa, Gozo Tsujimoto.
Application Number | 20080004227 10/591186 |
Document ID | / |
Family ID | 34914478 |
Filed Date | 2008-01-03 |
United States Patent
Application |
20080004227 |
Kind Code |
A1 |
Hirasawa; Akira ; et
al. |
January 3, 2008 |
Pharmaceutical Composition for Lowering Blood Sugar Level
Abstract
An object of the invention is to provide a pharmaceutical
composition and a kit therefor for the lowering of elevated blood
sugar level associated with diabetes and for the prevention of the
elevation of blood sugar level. The invention provides a
pharmaceutical composition containing a ligand (agonist or
antagonist) for a GT01 polypeptide, particularly a free fatty acid
as a GT01 polypeptide-specific ligand, or a GT01 gene or the GT01
polypeptide, for treating the hyperglycemia associated with
diabetes, and a kit therefor. The invention also provides a ligand
specific for the GT01 polypeptide, and a screening method exerting
an influence on the binding of the specific ligand to the GT01
polypeptide.
Inventors: |
Hirasawa; Akira; (Kyoto-fu,
JP) ; Tsujimoto; Gozo; (Tokyo, JP) |
Correspondence
Address: |
BACON & THOMAS, PLLC
625 SLATERS LANE
FOURTH FLOOR
ALEXANDRIA
VA
22314
US
|
Assignee: |
PHARMAFRONTIER CO., LTD
448-5 KAJII-CHO MIKURMANICHI-DORI , SEIWINGUCHI AGARU
HIGASHIGAWA KAMIKYO-KU
JAPAN
JP
6020841
|
Family ID: |
34914478 |
Appl. No.: |
10/591186 |
Filed: |
September 3, 2004 |
PCT Filed: |
September 3, 2004 |
PCT NO: |
PCT/JP04/12848 |
371 Date: |
April 23, 2007 |
Current U.S.
Class: |
514/44R ;
435/320.1; 436/501; 530/350; 536/23.1 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
3/10 20180101; G01N 33/74 20130101; G01N 2333/726 20130101; G01N
2800/042 20130101; G01N 2500/00 20130101; A61P 1/14 20180101; A61P
43/00 20180101; C07K 14/723 20130101; C07K 14/605 20130101; C07K
14/705 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/044 ;
435/320.1; 436/501; 530/350; 536/023.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61P 43/00 20060101 A61P043/00; C07H 21/04 20060101
C07H021/04; C07K 14/00 20060101 C07K014/00; C12N 15/00 20060101
C12N015/00; G01N 33/566 20060101 G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 2004 |
JP |
2004-056452 |
Aug 20, 2004 |
JP |
2004-240607 |
Claims
1. A polypeptide, or a salt thereof, comprising the amino acid
sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and having the
activity of promoting GLP-1 secretion from a cell.
2. A polypeptide, or a salt thereof, comprising an amino acid
sequence formed by providing deletion, substitution, or addition of
one or several amino acids into the amino acid sequence represented
by SEQ ID NO: 1 or SEQ ID NO: 2, and having the activity of
promoting GLP-1 secretion from a cell.
3. A polypeptide, or a salt thereof, comprising an amino acid
sequence having at least 85% sequence identity to the amino acid
sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and having the
activity of promoting GLP-1 secretion from a cell.
4. The polypeptide or a salt thereof according to any one of claims
1 to 3, wherein GLP-1 induces insulin secretion.
5. The polypeptide or a salt thereof according to any one of claims
1 to 4, present on an enteroendocrine cell.
6. A polynucleotide containing a polynucleotide encoding the
polypeptide according to any one of claims 1 to 5.
7. A recombinant vector containing the polynucleotide according to
claim 6.
8. A pharmaceutical composition for lowering the high blood sugar
level associated with diabetes, containing the vector according to
claim 7 as an active ingredient.
9. The pharmaceutical composition according to claim 8, for
inducing insulin secretion.
10. A ligand for the polypeptide or a salt thereof according to any
one of claims 1 to 5.
11. The ligand according to claim 10, wherein the ligand is a
straight-chain or branched free fatty acid.
12. The ligand according to claim 11, wherein said free fatty acid
has a carbon number of 10 to 24.
13. The ligand according to claim 11 or 12, wherein said free fatty
acid has an unsaturated bond number of 0 to 6.
14. The ligand according to claim 13, wherein said free fatty acid
is selected from the group consisting of capric acid, lauric acid,
myristic acid, pentadecanoic acid, palmitic acid, stearic acid,
arachic acid, behenic acid, margaric acid, palmitoleic acid,
eicosatrienoic acid, elaidic acid, petroselinic acid, oleic acid,
.alpha.-linolenic acid, .gamma.-linolenic acid,
homo-.gamma.-linolenic acid, arachidonic acid, eicosadienoic acid,
eicosatrienoic acid, eicosapentaenoic acid, docosahexaenoic acid,
linoleic acid, eicosatetraenoic acid, and vaccenic acid.
15. A pharmaceutical composition for lowering the high blood sugar
level associated with diabetes, containing the ligand according to
any one of claims 10 to 14 as an active ingredient.
16. The pharmaceutical composition according to claim 15, for
inducing insulin secretion.
17. A method for determining a ligand for the polypeptide or a salt
thereof according to any one of claims 1 to 5, the method
comprising the step of examining the specific binding ability of a
candidate ligand substance to the polypeptide or a salt
thereof.
18. A method for screening a substance altering the binding
properties of a ligand for the polypeptide or a salt thereof
according to any one of claims 1 to 5 to the polypeptide or a salt
thereof, the method comprising using the polypeptide or a salt
thereof.
19. A kit for screening a substance altering the binding properties
of a ligand for the polypeptide or a salt thereof according to any
one of claims 1 to 5 to the polypeptide or a salt thereof, which
contains the polypeptide or a salt thereof as a constituent.
20. A method for detecting the polypeptide or a salt thereof
according to any of claims 1 to 5 in a sample derived from a
subject suffering from or at risk of diabetes, which comprises
contacting the sample with an agent specifically recognizing the
polypeptide or a salt thereof to detect the polypeptide or a salt
thereof.
21. The method according to claim 20, wherein said agent is an
antibody capable of specifically binding to the polypeptide or a
salt thereof according to any of claims 1 to 5.
22. A kit used in the method according to claim 20 or 21, which
contains said agent as an essential constituent.
23. A polypeptide, or a salt thereof, comprising the amino acid
sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and having the
activity of promoting CCK secretion from a cell.
24. A polypeptide, or a salt thereof, comprising an amino acid
sequence formed by providing deletion, substitution, or addition of
one or several amino acids into the amino acid sequence represented
by SEQ ID NO: 1 or SEQ ID NO: 2, and having the activity of
promoting CCK secretion from a cell.
25. A polypeptide, or a salt thereof, comprising an amino acid
sequence having at least 85% sequence identity to the amino acid
sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and having the
activity of promoting CCK secretion from a cell.
26. The polypeptide or a salt thereof according to any one of
claims 23 to 25, present on an enteroendocrine cell.
27. A polynucleotide containing a polynucleotide encoding the
polypeptide according to any one of claims 23 to 26.
28. A recombinant vector containing the polynucleotide according to
claim 27.
29. A pharmaceutical composition for treating eating disorders,
containing the vector according to claim 28 as an active
ingredient.
30. A ligand for the polypeptide or a salt thereof according to any
one of claims 23 to 26.
31. The ligand according to claim 30, wherein the ligand is a
straight-chain or branched free fatty acid.
32. The ligand according to claim 31, wherein said free fatty acid
has a carbon number of 10 to 24.
33. The ligand according to claim 31 or 32, wherein said free fatty
acid has an unsaturated bond number of 0 to 6.
34. The ligand according to claim 33, wherein said free fatty acid
is selected from the group consisting of capric acid, lauric acid,
myristic acid, pentadecanoic acid, palmitic acid, stearic acid,
arachic acid, behenic acid, margaric acid, palmitoleic acid,
eicosatrienoic acid, elaidic acid, petroselinic acid, oleic acid,
.alpha.-linolenic acid, .gamma.-linolenic acid,
homo-.gamma.-linolenic acid, arachidonic acid, eicosadienoic acid,
eicosatrienoic acid, eicosapentaenoic acid, docosahexaenoic acid,
linoleic acid, eicosatetraenoic acid, and vaccenic acid.
35. A pharmaceutical composition for treating eating disorders,
containing the ligand according to anyone of claims 30 to 34 as an
active ingredient.
36. The pharmaceutical composition according to claim 29 or 35, for
treating obesity.
37. The pharmaceutical composition according to claim 29 or 35, for
treating cibophobia.
38. A dietary supplement composition containing the ligand
according to any one of claims 30 to 34 as an active
ingredient.
39. The dietary supplement composition according to claim 38, used
for rational dieting.
40. The dietary supplement composition according to claim 38, used
for alleviating anorexia.
41. A method for determining a ligand for the polypeptide or a salt
thereof according to any one of claims 23 to 26, the method
comprising the step of examining the specific binding ability of a
candidate ligand substance to the polypeptide or a salt
thereof.
42. A method for screening a substance altering the binding
properties of a ligand for the polypeptide or a salt thereof
according to any one of claims 23 to 26 to the polypeptide or a
salt thereof, the method comprising using the polypeptide or a salt
thereof.
43. A kit for screening a substance altering the binding properties
of a ligand for the polypeptide or a salt thereof according to any
one of claims 23 to 26 to the polypeptide or a salt thereof, which
contains the polypeptide or a salt thereof as a constituent.
44. A method for detecting the polypeptide or a salt thereof
according to any of claims 23 to 26 in a sample derived from a
subject suffering from or at risk of an eating disorder, which
comprises contacting the sample with an agent specifically
recognizing the polypeptide or a salt thereof to detect the
polypeptide or a salt thereof.
45. The method according to claim 44, wherein said agent is an
antibody capable of specifically binding to the polypeptide or a
salt thereof according to any of claims 23 to 26.
46. A kit used in the method according to claim 44 or 45, which
contains said agent as an essential constituent.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition for lowering blood sugar level and preventing or
treating diabetes.
[0002] In addition, the present invention relates to a
pharmaceutical composition for treating eating disorders.
[0003] Further, the present invention relates to a dietary
supplement composition used for rational dieting.
BACKGROUND ART
[0004] The number of diabetic patients is markedly increased in
society today, and social and economical costs considered necessary
for the treatment thereof have reached a large amount of money,
which provides a serious problem in the medical field desired to be
early solved.
[0005] Diabetes does not enable glucose in blood to be normally
metabolized, makes a so-called hyperglycemic state persist, and has
various negative effects on the function of the body. Major
complications of diabetes include retinopathy, nephropathy,
neuropathy, and arteriosclerosis, and these complications have a
risk causing serious pathologic conditions such as blindness,
uremia, stroke, and cardiac infarction.
[0006] As a method for treating diabetes, the injection of insulin
necessary for glucose metabolism is conducted in addition to
dietetic therapy, ergotherapy, or the like to normalize metabolic
function.
[0007] In addition to the method involving the direct
administration of insulin, researches, for example, on a method for
promoting insulin production, insulin secretion, or the like to
attain the lowering of blood sugar level are also actively carried
out.
[0008] GLP-1 (glucagon-like peptide-1) is getting a lot of
attention as a peptide hormone regulating insulin secretion. GLP-1
is a peptide hormone arising from the post-translation processing
of proglucagon produced in L cells which are enteroendocrine cells
present in the ileum, large intestine, or the like (Drucker, 1998).
Since it induces insulin secretion in response to a glucose
concentration, the peptide has been pointed out to have a
possibility of exerting a therapeutic effect particularly against
type II (non-insulin-dependent) diabetes (Drucker, 2001; Thorens
and Waeber, 1993). In addition, GLP-1 has been reported to have,
for example, the effect of inducing the proliferation of
.beta.-cells or the neogenesis of .beta.-cells from stem cells, and
is probably effective in retarding the apoptosis of .beta.-cells in
type II diabetes and in maintaining the effect of pancreatic islet
transplantation against type I diabetes.
[0009] In addition to the effect of regulating insulin secretion,
GLP-1 has been shown to have the effect of inducing the stimulation
of "feeling of satiety" and the reduction of eating by acting on
the hypothalamus in the central nervous system (Turton, et al.,
1996; Flint, et al., 1998); it is expected to have the therapeutic
effects not only against diabetes but also against eating disorders
including obesity. Recent studies have shown that substances such
as fatty acid and TNF released from fat cells disturb insulin
action, and a mechanism through which the elevation of blood sugar
level due to obesity is promoted is also being elucidated. Thus,
GLP-1 not only promoting insulin secretion but also simultaneously
having the effect of suppressing obesity will provide a
breakthrough therapeutic agent for treating diabetes.
[0010] In applying GLP-1 to the treatment of diabetes, however,
some trouble has been caused e.g., by the points that the half-life
of GLP-1 is short in the living body (Drucker, 2001; Thorens and
Waeber, 1993) and that a method by injection has to be adopted in
the administration thereof because it is peptidic.
[0011] A possible method for overcoming these problems is to
regulate insulin secretion by promoting the secretion of endogenous
GLP-1.
[0012] It has been previously pointed out that fatty acid is
involved in the promotion of GLP-1 secretion. For example, it has
been reported that giving a monounsaturated fatty acid to rats
stimulates the secretion of GLP-1 (non-patent document 1) or that
oleic acid or palmitic acid promotes the secretion of GLP-1 from
the human intestinal cell line NCI-H716 (non-patent document 2).
Meanwhile, however, there have been reported some results
inconsistent with the above-mentioned reports, including that a
polyunsaturated fatty acid or monounsaturated fatty acid did not
affect insulin secretion via GLP-1 (non-patent document 3) and that
the injection of a short-chain fatty acid (SCFA) into blood
produced no change in the concentration of GLP-1 in plasma
(non-patent document 4). In the case of the reported promotion of
GLP-1 secretion by certain types of fatty acids, further, no
mechanism of action thereof has been elucidated, which has also
brought into question the validity of relation between fatty acid
and GLP-1 promotion.
[0013] The composition according to the invention contains a GT01
protein as a G-protein coupled receptor and a ligand specifically
binding to the GT01 protein. The G-protein coupled receptor is
called a 7-pass transmembrane receptor (7TMR) because it has 7
transmembrane regions (see FIG. 1), and involved in intracellular
signaling through the activation of a coupled guanine
nucleotide-binding protein (hereinafter referred to as G
protein).
[0014] The G-protein coupled receptor is present on the surface of
each functional cell in the living body, forms a target for a
ligand molecule regulating the function, and conveys a signal into
the cell through the binding with the ligand molecule. The cell
receiving the conveyed signal is subjected to the activation or
inhibition of the cellular function, resulting in the initiation of
various in vivo reactions. Thus, elucidating the function of the
G-protein coupled receptor has become highly important also in view
of developing a medicine well regulating an in vivo reaction.
[0015] A vast amount of information of genome and cDNA has become
available in recent years, and many G-protein coupled receptors
have been identified; however, in many of those, the functions
thereof and specific ligands therefor have not yet been
demonstrated, the progress of analysis thereof having been
awaited.
[0016] Human GT01 protein is called GPR120, and identical to the
amino acid sequence deposited as NP.sub.--859529 in GenBank.
However, no function of GPR120 has been addressed, and the
biological role thereof is uncertain (non-patent document 5).
[0017] In addition, it has 95% amino acid identity to a 14273
receptor as a G-protein coupled receptor. However, no ligand for
the 14273 receptor has been identified, and the mechanism of action
thereof has not been elucidated in detail. The 14273 receptor has
been also observed to be expressed in the heart, and identified as
an involvement in heart disease from the analysis of transgenic
mice using a gene encoding the receptor. Thus, although the GT01
polypeptide disclosed in the invention and the 14273 receptor have
a high amino acid sequence identity with each other, from the
comparison of the functions thereof (to be described), it seems
that they have the possibility of assuming physiologically
completely different roles (see patent document 1 or 2).
[0018] The present inventors have already found that the GT01
protein according to the invention, together with a ligand
therefor, acts to promote the secretion of CCK functioning in the
control of eating (Japanese Patent Application No. 2003-180375).
The present invention has additionally found that the GT01 protein,
together with an agonist therefor, acts to promote the secretion of
GLP-1 functioning in the lowering of blood sugar level and further
the secretion of insulin. Thus, it is expected that the GT01
protein according to the invention, together with a ligand
therefor, prevents or alleviates obesity by enabling the control of
eating and the lowering of blood sugar level and also exerts an
effect in the treatment of diabetes by producing the lowering of
blood sugar level.
[0019] Patent document 1: U.S. Pat. No. 6,448,005B1 (entire
text).
[0020] Patent document 2: National Publication of International
Patent Application No. 2002-536997 (entire text).
[0021] Non-patent document 1: Rocca, A. S. et al., Endocrinology
142:1148-1155, 2001.
[0022] Non-patent document 2: Reimer, R. A. et al., Endocrinoloby
142:4522-4528, 2001.
[0023] Non-patent document 3: Cuche, G. et al., Am J Physiol
Gastrointest Liver Physiol. 279: G925-930, 2000.
[0024] Non-patent document 4: Brynes, A. E., Am J Clin Nutr 72:
1111-1118, 2000.
DISCLOSURE OF THE INVENTION
[0025] Non-patent document 5: Fredriksson, R. et al., FEBS 554:
381-388, 2003.
[0026] Then, an object of the present invention is to provide a
pharmaceutical composition for inducing the secretion of endogenous
GLP-1 useful in treating type I or type II diabetes by promoting
insulin secretion.
[0027] Another object of the invention is to provide a method for
identifying a ligand for a GT01 protein suitable for GLP-1
secretion and insulin secretion, a screening method for obtaining a
compound altering the binding properties of the ligand to the GT01
protein, and a kit for use in the screening.
[0028] In view of the above-described circumstances, as the result
of intensive studies for analyzing the function of a GT01
polypeptide (for example, SEQ ID NO: 1 or2) and identifying a
compound serving as a ligand therefor, the present inventors have
first demonstrated here that the GT01 polypeptide is distributed on
the surface of human enteroendocrine cells and has the function of
promoting the secretion of GLP-1 functioning in the control of
insulin secretion, and further insulin secretion is actually
promoted, and also has revealed a compound serving as a ligand for
the GT01 polypeptide.
[0029] In this respect, the terms "polypeptide" and "protein" shall
be used interchangeably herein unless otherwise annotated.
[0030] Thus, the above-described problems are solved by terms (1)
to (40) below.
[0031] (1) An embodiment according to a first invention of the
present invention is "a polypeptide, or a salt thereof, comprising
the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2
and having the activity of promoting GLP-1 secretion from a
cell".
[0032] Here, "cell" is a mammal cell unless otherwise annotated,
and particularly refers to, but not limited to, for example, a cell
present in the living body and having the ability to release GLP-1
(for example, an L cell as an enteroendocrine cell) or an
established cell line having the ability to release GLP-1 in vitro
(for example, an STC-1 cell, NCI-H716 cell, or GLUTag cell)
(hereinafter referred to the same).
[0033] (2) An embodiment according to a second invention of the
present invention is "a polypeptide, or a salt thereof, comprising
an amino acid sequence formed by providing deletion, substitution,
or addition of one or several amino acids into the amino acid
sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and having
the activity of promoting GLP-1 secretion from a cell".
[0034] (3) An embodiment according to a third invention of the
present invention is "a polypeptide, or a salt thereof, comprising
an amino acid sequence having at least 85% sequence identity to the
amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and
having the activity of promoting GLP-1 secretion from a cell".
[0035] Here, "sequence identity" refers to the percentage of
identical amino acids based on a amino acid sequence to be compared
(here, for example, SEQ ID NO: 1 or SEQ ID NO: 2) when an amino
acid sequence to compare is aligned with the amino acid sequence to
be compared by introducing a gap or the like so as to maximize the
rate of amino acid concordance with the amino acid sequence to be
compared.
[0036] When the amino acid sequence of SEQ ID: 1 was aligned with
the amino acid sequence of SEQ ID: 2 by introducing a gap or the
like so as to maximize the rate of amino acid concordance between
these amino acid sequences, 83% of the amino acid sequence of SEQ
ID: 1 were identical with the amino acid sequence of SEQ ID: 2.
Since SEQ ID: 1 and SEQ ID: 2 represent the amino acid sequences of
human GT01 and mouse GT01, respectively, a polypeptide having at
least 83% sequence identity with each of the amino acid sequences
is predicted to be a GT01 homolog in another organism species.
[0037] (4) An embodiment according to a fourth invention of the
present invention is "the polypeptide or a salt thereof described
in any of terms (1) to (3) above, wherein the above-described GLP-1
induces insulin secretion".
[0038] (5) An embodiment according to a fifth invention of the
present invention is "the polypeptide or a salt thereof described
in any of terms (1) to (4) above, present on an enteroendocrine
cell".
[0039] (6) An embodiment according to a sixth invention of the
present invention is "a polynucleotide containing a polynucleotide
encoding the polypeptide described in any of terms (1) to (5)
above".
[0040] (7) An embodiment according to a seventh invention of the
present invention is "a recombinant vector containing the
polynucleotide described in term (6) above".
[0041] (8) An embodiment according to an eighth invention of the
present invention is "a pharmaceutical composition for lowering the
high blood sugar level associated with diabetes, containing the
vector described in term (7) above as an active ingredient".
[0042] (9) An embodiment according to a ninth invention of the
present invention is "the pharmaceutical composition described in
term (8) above, for inducing insulin secretion".
[0043] (10) An embodiment according to a tenth invention of the
present invention is "a ligand for the polypeptide or a salt
thereof described in any of terms (1) to (5) above".
[0044] (11) An embodiment according to an eleventh invention of the
present invention is "the ligand described in term (10) above,
wherein the ligand is a straight-chain or branched free fatty
acid".
[0045] (12) An embodiment according to a twelfth invention of the
present invention is "the ligand described in term (11) above,
wherein the above-described free fatty acid has a carbon number of
10 to 24".
[0046] (13) An embodiment according to a thirteenth invention of
the present invention is "the ligand described in term (11) or (12)
above, wherein the above-described free fatty acid has an
unsaturated bond number of 0 to 6".
[0047] (14) An embodiment according to a fourteenth invention of
the present invention is "the ligand described in term (13) above,
wherein the above-described free fatty acid is selected from the
group consisting of capric acid, lauric acid, myristic acid,
pentadecanoic acid, palmitic acid, stearic acid, arachic acid,
behenic acid, margaric acid, palmitoleic acid, eicosatrienoic acid,
elaidic acid, petroselinic acid, oleic acid, .alpha.-linolenic
acid, .gamma.-linolenic acid, homo-.gamma.-linolenic acid,
arachidonic acid, eicosadienoic acid, eicosatrienoic acid,
eicosapentaenoic acid, docosahexaenoic acid, linoleic acid,
eicosatetraenoic acid, and vaccenic acid".
[0048] Here, when isomers are present for the fatty acid described
in term (14) above, all of the isomers shall be included; however,
for example, it is particularly preferable that eicosatrienoic acid
is cis-5,8,11-eicosatrienoic acid; eicosadienoic acid is
cis-11,14-eicosadienoic acid; eicosatrienoic acid is
cis-11,14,17-eicosatrienoic acid; eicosatetraenoic acid is
cis-7,10,13,16-eicosatetraenoic acid; eicosapentaenoic acid is
cis-5,8,11,14,17-eicosapentaenoic acid or
all-cis-7,10,13,16,19-eicosapentaenoic acid; and docosahexaenoic
acid is cis-4,7,10,13,16,19-docosahexaenoic acid.
[0049] (15) An embodiment according to a fifteenth invention of the
present invention is "a pharmaceutical composition for lowering the
high blood sugar level associated with diabetes, containing the
ligand described in any of terms (10) to (14) above as an active
ingredient".
[0050] (16) An embodiment according to a sixteenth invention of the
present invention is "the pharmaceutical composition described in
term (15) above, for inducing insulin secretion".
[0051] (17) An embodiment according to a seventeenth invention of
the present invention is "a method for determining a ligand for the
polypeptide or a salt thereof described in any of terms (1) to (5)
above, characterized by comprising the step of examining the
specific binding ability of a candidate ligand substance to the
polypeptide or a salt thereof".
[0052] (18) An embodiment according to an eighteenth invention of
the present invention is "a method for screening a substance
altering the binding properties of a ligand for the polypeptide or
a salt thereof described in any of terms (1) to (5) above to the
polypeptide or a salt thereof, characterized by using the
polypeptide or a salt thereof".
[0053] Here, "substance altering the binding properties" includes a
substance acting so as to enhance the binding properties and a
substance acting so as to reduce the binding properties.
[0054] (19) An embodiment according to a nineteenth invention of
the present invention is "a kit for screening a substance altering
the binding properties of a ligand for the polypeptide or a salt
thereof described in any of terms (1) to (5) above to the
polypeptide or a salt thereof, characterized by containing the
polypeptide or a salt thereof as a constituent".
[0055] (20) An embodiment according to a twentieth invention of the
present invention is "a method for detecting the polypeptide or a
salt thereof described in any of terms (1) to (5) above in a sample
derived from a subject suffering from or at risk of diabetes,
characterized by contacting the sample with an agent specifically
recognizing the polypeptide or a salt thereof to detect the
polypeptide or a salt thereof".
[0056] (21) An embodiment according to a twenty-first invention of
the present invention is "the method described in term (20) above,
characterized in that the above-described agent is an antibody
capable of specifically binding to the polypeptide or a salt
thereof described in any of terms (1) to (5) above".
[0057] (22) An embodiment according to a twenty-second invention of
the present invention is "a kit used in the method described in
term (20) or (21) above, characterized by containing the
above-described agent as an essential constituent".
[0058] (23) An embodiment according to a twenty-third invention of
the present invention is "a polypeptide, or a salt thereof,
comprising the amino acid sequence represented by SEQ ID NO: 1 or
SEQ ID NO: 2 and having the activity of promoting CCK secretion
from a cell".
[0059] Here, "cell" is a mammal cell unless otherwise annotated,
and particularly refers to, but not limited to, for example, a cell
present in the living body and having the ability to release CCK
(for example, an L cell as an enteroendocrine cell) or an
established cell line having the ability to release GLP-1 in vitro
(for example, an STC-1 cell, NCI-H716 cell, or GLUTag cell)
(hereinafter referred to the same).
[0060] (24) An embodiment according to a twenty-fourth invention of
the present invention is "a polypeptide, or a salt thereof,
comprising an amino acid sequence formed by providing deletion,
substitution, or addition of one or several amino acids into the
amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2,
and having the activity of promoting CCK secretion from a
cell".
[0061] (25) An embodiment according to a twenty-fifth invention of
the present invention is "a polypeptide, or a salt thereof,
comprising an amino acid sequence having at least 85% sequence
identity to the amino acid sequence represented by SEQ ID NO: 1 or
SEQ ID NO: 2 and having the activity of promoting CCK secretion
from a cell".
[0062] Here, "sequence identity" refers to the percentage of
identical amino acids based on a amino acid sequence to be compared
(here, for example, SEQ ID NO: 1 or SEQ ID NO: 2) when a amino acid
sequence to compare is aligned with the amino acid sequence to be
compared by introducing a gap or the like so as to maximize the
rate of amino acid concordance with the amino acid sequence to be
compared.
[0063] When the amino acid sequence of SEQ ID: 1 was aligned with
the amino acid sequence of SEQ ID: 2 by introducing a gap or the
like so as to maximize the rate of amino acid concordance between
these amino acid sequences, 83% of the amino acid sequence of SEQ
ID: 1 were identical with the amino acid sequence of SEQ ID: 2.
Since SEQ ID: 1 and SEQ ID: 2 represent the amino acid sequences of
human GT01 and mouse GT01, respectively, a polypeptide having at
least 83% sequence identity with each of the amino acid sequences
is predicted to be a GT01 homolog in another organism species.
[0064] (26) An embodiment according to a twenty-sixth invention of
the present invention is "the polypeptide or a salt thereof
described in any of terms (23) to (25) above, present on an
enteroendocrine cell".
[0065] (27) An embodiment according to a twenty-seventh invention
of the present invention is "a polynucleotide containing a
polynucleotide encoding the polypeptide described in any of terms
(23) to (26) above".
[0066] (28) An embodiment according to a twenty-eighth invention of
the present invention is "a recombinant vector containing the
polynucleotide described in term (27) above".
[0067] (29) An embodiment according to a twenty-ninth invention of
the present invention is "a pharmaceutical composition for treating
eating disorders, containing the vector described in term (28)
above as an active ingredient".
[0068] (30) An embodiment according to a thirtieth invention of the
present invention is "a ligand for the polypeptide or a salt
thereof described in any of terms (23) to (26) above".
[0069] (31) An embodiment according to a thirty-first invention of
the present invention is "the ligand described in term (30) above,
wherein the ligand is a straight-chain or branched free fatty
acid".
[0070] (32) An embodiment according to a thirty-second invention of
the present invention is "the ligand described in term (31) above,
wherein the above-described free fatty acid has a carbon number of
10 to 24".
[0071] (33) An embodiment according to a thirty-third invention of
the present invention is "the ligand described in term (31) or (32)
above, wherein the above-described free fatty acid has an
unsaturated bond number of 0 to 6".
[0072] (34) An embodiment according to a thirty-fourth invention of
the present invention is "the ligand described in term (33) above,
wherein the above-described free fatty acid is selected from the
group consisting of capric acid, lauric acid, myristic acid,
pentadecanoic acid, palmitic acid, stearic acid, arachic acid,
behenic acid, margaric acid, palmitoleic acid, eicosatrienoic acid,
elaidic acid, petroselinic acid, oleic acid, .alpha.-linolenic
acid, .gamma.-linolenic acid, homo-.gamma.-linolenic acid,
arachidonic acid, eicosadienoic acid, eicosatrienoic acid,
eicosapentaenoic acid, docosahexaenoic acid, linoleic acid,
eicosatetraenoic acid, and vaccenic acid".
[0073] Here, when isomers are present for the fatty acid described
in term (34) above, all of the isomers shall be included; however,
for example, it is particularly preferable that eicosatrienoic acid
is cis-5,8,11-eicosatrienoic acid; eicosadienoic acid is
cis-11,14-eicosadienoic acid; eicosatrienoic acid is
cis-11,14,17-eicosatrienoic acid; eicosatetraenoic acid is
cis-7,10,13,16-eicosatetraenoic acid; eicosapentaenoic acid is
cis-5,8,11,14,17-eicosapentaenoic acid or
all-cis-7,10,13,16,19-eicosapentaenoic acid; and docosahexaenoic
acid is cis-4,7,10,13,16,19-docosahexaenoic acid.
[0074] (35) An embodiment according to a thirty-fifth invention of
the present invention is "a pharmaceutical composition for treating
eating disorders, containing the ligand described in any of terms
(30) to (34) above as an active ingredient".
[0075] (36) An embodiment according to a thirty-sixth invention of
the present invention is "the pharmaceutical composition described
in term (35) above, for treating obesity".
[0076] (37) An embodiment according to a thirty-seventh invention
of the present invention is "the pharmaceutical composition
described in term (35) above, for treating cibophobia".
[0077] (38) An embodiment according to a thirty-eighth invention of
the present invention is "a dietary supplement composition
containing the ligand described in any of terms (30) to (34) above
as an active ingredient".
[0078] (39) An embodiment according to a thirty-ninth invention of
the present invention is "the dietary supplement composition
described in term (38) above, used for rational dieting".
[0079] (40) An embodiment according to a fortieth invention of the
present invention is "the dietary supplement composition described
in term (38) above, used for alleviating anorexia".
[0080] (41) An embodiment according to a forty-first invention of
the present invention is "a method for determining a ligand for the
polypeptide or a salt thereof described in any of terms (23) to
(26) above, characterized by comprising the step of examining the
specific binding ability of a candidate ligand substance to the
polypeptide or a salt thereof".
[0081] (42) An embodiment according to a forty-second invention of
the present invention is "a method for screening a substance
altering the binding properties of a ligand for the polypeptide or
a salt thereof described in any of terms (23) to (26) above to the
polypeptide or a salt thereof, characterized by using the
polypeptide or a salt thereof".
[0082] Here, "substance altering the binding properties" includes a
substance acting so as to enhance the binding properties and a
substance acting so as to reduce the binding properties.
[0083] (43) An embodiment according to a forty-third invention of
the present invention is "a kit for screening a substance altering
the binding properties of a ligand for the polypeptide or a salt
thereof described in any of terms (23) to (26) above to the
polypeptide or a salt thereof, characterized by containing the
polypeptide or a salt thereof as a constituent".
[0084] (44) An embodiment according to a forty-fourth invention of
the present invention is "a method for detecting the polypeptide or
a salt thereof described in any of terms (23) to (26) above in a
sample derived from a subject suffering from or at risk of an
eating disorder, characterized by comprising contacting the sample
with an agent specifically recognizing the polypeptide or a salt
thereof to detect the polypeptide or a salt thereof".
[0085] (45) An embodiment according to a forty-fifth invention of
the present invention is "the method described in term (44.) above,
characterized in that the above-described agent is an antibody
capable of specifically binding to the polypeptide or a salt there
of described in any of terms (23) to (26) above".
[0086] (46) An embodiment according to a forty-sixth invention of
the present invention is "a kit used in the method described in
term (44) or (45) above, characterized by containing the
above-described agent as an essential constituent".
[0087] The pharmaceutical composition provided by the invention can
be used to promote GLP-1 secretion and insulin secretion from
cells. As a result, there can be expected the effects of the
promoting of insulin secretion from pancreatic .beta.-cells, the
propagating of .beta.-cells, the inducing of the neogenesis of
.beta.-cells from stem cells, or the like.
[0088] In addition, there can be expected the effects of the
retarding of the apoptosis of .beta.-cells in type II diabetes and
the maintaining of the effect of pancreatic islet transplantation
against type I diabetes.
[0089] Further, the use of a pharmaceutical composition containing
a free fatty acid as a ligand for the GT01 polypeptide first
demonstrated by the invention enables the regulation of CCK release
from intestinal cells expressing the GT01 polypeptide. As a result,
the regulation of a peripheral or central eating-control mechanism
responsive to CCK is made possible, and the improvement of eating
disorders and symptoms of diseases etc. associated therewith can be
achieved.
[0090] Further, the accomplishment of rational dieting or the
effect of enhancing appetite can be expected by the ingestion of a
dietary supplement agent containing a free fatty acid as a ligand
for the GT01 polypeptide of the invention.
[0091] Since the pharmaceutical composition provided by the
invention is used to produce "feeling of satiety" to enable the
reduction of eating to be induced, it also suppresses obesity
probably facilitating diabetic symptoms and can be expected to be
further effective in the treatment of diabetes via the lowering of
blood sugar level.
[0092] Further, the method for determining a ligand or method for
screening a substance altering the binding properties of a ligand
to GT01, provided by the invention is used to enable the
identification of a new compound capable of effectively controlling
the secretion of GLP-1; the development of a therapeutic agent for
diabetes via the lowering of blood sugar level can be expected.
[0093] Further, the screening method or screening kit provided by
the invention is used to enable the identification of a new
compound capable of regulating the binding mode of an existing
ligand to GT01; the development of a therapeutic agent for diabetes
via the lowering of blood sugar level can be expected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] FIG. 1 is a scheme of a 7-pass transmembrane receptor;
[0095] FIG. 2 is an image showing the tissue specificity of a mouse
GT01 gene expression. GAPDH (glyceraldehydes-3-phosphate
dehydrogenase) was used as a control for comparing the
expression;
[0096] FIG. 3 is a fluorescence microscopic image showing that, as
a result of the binding of a ligand to a GT01 polypeptide, the
polypeptide migrates into the cell together with the ligand;
[0097] FIG. 4 is the results of determining the increase in
intracellular calcium concentration induced by a free fatty acid by
detecting fluorescence intensity at 510 to 570 nm for excitation
light of 488 nm using FLIPR (Fluorometric Imaging Plate Reader,
Molecular Devices);
[0098] FIG. 5 is a diagram showing pEC.sub.50s for increases in the
concentration of Ca.sup.2+ in an HEK cell when various free fatty
acids were added;
[0099] FIG. 6 is a set of graphs showing an effect of an antisense
for human GT01 on the increase in intracellular calcium
concentration induced by .alpha.-linolenic acid;
[0100] FIG. 7 is a graph showing the free fatty acid concentration
dependency of the CCK release induced by various free fatty
acids;
[0101] FIG. 8 is a graph showing the amounts of the GLP-1 release
(ordinate) induced by various fatty acids (.alpha.-linolenic acid,
docosahexaenoic acid (DHA), palmitoleic acid, oleic acid, stearic
acid, octanoic acid, and .alpha.-linolenic acid-methyl ester). The
fatty acids are each used in concentrations of 10 .mu.M, 3 .mu.M,
and 100 .mu.M (abscissa);
[0102] FIG. 9 is the results of in situ hybridization regarding
GT01 and GLP-1 in mouse colon;
[0103] FIG. 10 is the results of in situ hybridization regarding
GT01 and GLP-1 in human colon;
[0104] FIG. 11 is a graph showing the variation of GLP-1
concentration in mouse portal vein by giving various fatty acids
(.alpha.-linolenic acid, stearic acid, and octanoic acid) to the
mice. For negative controls, the determination of concentration was
carried out when neither fatty acid nor vehicle were given
(control) and when only vehicle was given (vehicle);
[0105] FIG. 12 is a graph showing the concentration of GLP-1 in
mouse portal vein or inferior vena cava 0.5 hr or 2 hrs after
giving .alpha.-linolenic acid (.alpha.-LA) to the mice. For
negative controls, the determination of concentration was carried
out when neither fatty acid nor vehicle were given (control) and
when only vehicle was given (vehicle); and
[0106] FIG. 13 is a graph showing the concentration of insulin in
mouse portal vein or inferior vena cava 0.5 hr or 2 hrs after
giving .alpha.-linolenic acid (.alpha.-LA) to the mice. For
negative controls, the determination of concentration was carried
out when neither fatty acid nor vehicle was given (control) and
when only vehicle was given (vehicle).
BEST MODE FOR CARRYING OUT THE INVENTION
1. Cloning of a Gene Encoding a GT01 Polypeptide
[0107] For the purpose of the invention, "GT01 polypeptide" refers
to a G-protein coupled receptor having the amino acid sequence
represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a G-protein coupled
receptor which consists of the amino acid sequence represented by
SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence has
deletion, substitution, or addition of one or several amino acids,
is distributed on the surface of an enteroendocrine cell (for
example, an L cell) or an enteroendocrine cell line (for example,
STC-1 or NCI-H716 cell or GLUTag cell line), and binds to a ligand
therefor to convey, into a cell, a signal for GLP-1 secretion.
[0108] The GT01 polypeptide gene according to the invention may be
cloned from, for example, a cDNA library or a genomic DNA library
using PCR primers prepared based on the sequence information
deposited in a public database (NCBI) (accession number:
NM.sub.--181748 (mouse), NM.sub.--181745 (human), etc.).
[0109] The PCR primer can be designed using a primer design
software program such as Primer 3 (Whitehead Institute for
Biomedical Research). In addition, the PCR primer can be
synthesized using a standard synthesis technique employing, for
example, an automatic DNA synthesizer, but maybe obtained from a
commercial source. The amplified product expected to result from a
PCR reaction preferably has such a length that amplification is
efficient and subsequent separation by agarose gel and base
sequence analysis are easy, and the design is preferably performed
so that it has a length of, for example, 80 to 200 bases. The PCR
reaction is carried out employing the prepared PCR primer and using
a cDNA library or the like as a template, and it is confirmed e.g.,
by sequencing that the amplified product obtained is a desired
product.
[0110] The cDNA library used here may be prepared from cells of any
animal including human (for example, human, mouse, rat, guinea pig,
chick, rabbit, pig, cattle, monkey, sheep, dog, or cat), including,
but not limited to, immune system cells, hematocytic cells,
fibroblasts, splenocytes, hepatocytes, marrow cells, pancreatic
cells, Langerhans cells, epithelial cells, muscle cells, nerve
cells, glia cells, fat cells, or established cell lines thereof or
precursor cells thereof, or may be prepared from a tissue of any
animal (for example, human, mouse, rat, guinea pig, chick, rabbit,
pig, cattle, monkey, sheep, dog, or cat), including, but not
limited to, brain, spinal cord, pituitary gland, thymus, peripheral
blood, spleen, lymphatic tissue, pituitary gland, stomach,
pancreas, kidney, genital gland, thyroid gland, gall bladder,
testis, orchis, ovary, placenta, uterus, bone, joint, or skeletal
muscle. The preparation of the cDNA library may be carried out
using a conventional technique in the art (see, for example,
Sambrook, et al., 1989).
2. A Ligand for the GT01 Polypeptide
[0111] As used herein, "ligand" refers to a chemical molecule
fitting a receptor, and includes both concepts of "agonist" and
"antagonist".
[0112] In addition, "agonist" as used herein includes any of the
molecules inducing the biological activities of an endogenous GT01
polypeptide (the activities of binding to a ligand to convey, into
a cell, a signal for GLP-1 secretion). On the other hand,
"antagonist" includes any of the molecules competing with an
agonist to inhibit and neutralize part or all of the biological
activities of an endogenous GT01 polypeptide. According to the
invention, "agonist" may particularly have, but not limited to, the
effect of promoting GLP-1 secretion.
[0113] (1) Identification of a Ligand
[0114] When a compound such as the one to be tested is added to an
assay system for detecting the biological activities of a GT01
polypeptide (the activities of binding to a ligand to convey, into
a cell, a signal for GLP-1 secretion), the compound is an agonist
if the biological activities of the GT01 polypeptide are promoted,
and conversely, the compound is an antagonist if the activities are
suppressed. Specifically, by way of non-limiting example, when, in
the determination of intracellular calcium concentration shown in
Examples to be described, a test compound is allowed to contact
with a cell used in the assay, it can be deemed that the test
compound is an agonist if the intracellular calcium concentration
is increased and the compound is an antagonist if the increase of
the intracellular calcium concentration is inhibited even in the
presence of an agonist.
[0115] (2) Method for Identifying a Ligand
[0116] The method for determining a ligand according to the
invention is characterized in that there are determined the binding
amount of a candidate substance (test substance) to the GT01
protein of the invention or a partial peptide thereof, the
cell-stimulating activities thereof, and the like when the GT01
protein or the partial peptide is allowed to contact with the
candidate substance. More specifically, the invention provides: (i)
a method for identifying a ligand for the GT01 protein or a salt
thereof, characterized by comprising determining the binding amount
of a labeled candidate substance to the GT01 protein or a salt
thereof or a partial peptide of GT01 or a salt thereof when the
labeled test compound is allowed to contact with the GT01 protein
or a salt thereof or a partial peptide of GT01 or a salt thereof;
(ii) a method for identifying a ligand for the GT01 protein,
characterized by comprising determining the binding amount of a
labeled candidate substance to a cell containing the GT01 protein
or a membrane fraction of the cell when the labeled candidate
substance is allowed to contact with the cell or the membrane
fraction; (iii) a method for identifying a ligand for the GT01
protein or a salt thereof, characterized by comprising determining
the binding amount of a labeled candidate substance to the GT01
protein or a salt thereof when the labeled candidate substance is
allowed to contact with the GT01 protein expressed, by culturing a
transformant containing DNA encoding the GT01 protein, on the cell
membrane; (iv) a method for identifying a ligand for the GT01
protein or a salt thereof, characterized by comprising determining
the activities of stimulating a cell containing the GT01 protein
(for example, intracellular calcium concentration) via the GT01
protein when a candidate substance is allowed to contact with the
cell; and (v) a method for identifying a ligand for the GT01
protein of the invention or a salt thereof, characterized by
comprising determining cell-stimulating activities (for example,
intracellular calcium release, intracellular cAMP formation,
intracellular cGMP formation, inositol phosphate production, cell
membrane potential fluctuation, and intracellular protein
phosphorylation) via a receptor protein when a candidate substance
is allowed to contact with the receptor protein expressed, by
culturing the transformant containing DNA encoding GT01, on the
cell membrane.
[0117] The GT01 protein used in the method for identifying a ligand
according to the invention maybe any protein containing a GT01
protein or a partial peptide thereof, but is particularly
preferably a GT01 protein abundantly expressed using animal
cells.
[0118] (3) Antisense RNA or DNA
[0119] An antisense RNA or DNA against a GT01 gene has the
possibility of acting as an effective antagonist. The antisense RNA
or DNA molecule hybridizes with a target mRNA for inhibiting
translation to inhibit the function of a target factor. The
antisense RNA is, for example, designed so as to hybridize with
mRNA in vivo to inhibit translation from the mRNA into a GT01
polypeptide (Okano, et al., 1991). The DNA oligonucleotide is
designed, for example, so as to be complementary to the
transcription initiation region of the GT01 gene, resulting in the
inhibition of GT01 expression (Cohen, 1989).
[0120] The antisense RNA or DNA can be introduced into a cell in
such a way that they can be expressed in vivo so as to inhibit the
expression of the GT01 polypeptide. When the antisense DNA is used,
it is preferably, for example, an oligonucleotide binding to a
position falling within the range of about -10 to +10 bases from a
target gene sequence.
[0121] (4) Anti-GT01 Polypeptide Antibody
[0122] The invention includes an antibody capable of specifically
binding to the GT01 polypeptide, and a fragment thereof such as Fab
or (Fab).sub.2.
[0123] As used herein, "antibody" (anti-GT01; including an agonist,
antagonist, or neutralizing antibody) includes a
monoepitope-specific anti-GT01 polypeptide antibody, a
polyepitope-specific anti-GT01 polypeptide antibody, or a single
chain antibody thereof, or a fragment thereof.
[0124] These antibodies include, for example, a monoclonal
antibody, a polyclonal antibody, and a humanized antibody.
[0125] (4)-1. Polyclonal Antibody
[0126] The polyclonal antibody can be prepared, for example, by
injecting a mixture of an immunogen and an adjuvant into a mammal
host animal. Typically, the immunogen and/or adjuvant are
subcutaneously or intraperitoneally injected plural times to the
host animal. Examples of the immunogen include fusions of the GT01
polypeptide to polypeptides heterologous thereto, or fragments
thereof. Examples of the adjuvant include Freund's complete
adjuvant and monophosphoryl lipid A-synthetic trehalose
dicorynomicolate (MPL-TDM). To enhance an immune response, the
immunogen may be injected after binding to a protein having
immunogenicity such as keyhole limpet heamocyanin (KLH), serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
[0127] Alternatively, the preparation may be carried out using
chicken producing an IgY molecule (Schade, et al., 1996).
[0128] For details regarding methods for producing antibodies, see,
for example, Ausubel, et al., 1987 or Harlow and Lane, 1988.
[0129] (4)-2. Monoclonal Antibody
[0130] The anti-GT01 polypeptide monoclonal antibody is prepared
using a hybridoma method (Milstein and Cuello, 1983).
[0131] This method comprises the following 4 steps: (i) immunizing
a host animal or lymphocytes derived from the host animal; (ii)
recovering lymphocytes secreting (or potentially secreting)
monoclonal antibodies; (iii) fusing the lymphocytes to immortalized
cells; and (iv) selecting cells secreting a desired monoclonal
antibody (anti-GT01 polypeptide).
[0132] A mouse, rat, guinea pig, hamster, or another suitable host
animal is selected as an animal for immunization and injected with
an immunogen. Alternatively, lymphocytes obtained from the animal
for immunization may be immunized in vitro. When human cells are
desirable, peripheral blood lymphocytes (PBLs) are generally used.
However, spleen cells or lymphocytes derived from other mammal
animals are more common and preferable. Examples of the immunogen
include GT01 polypeptide and fusions of the GT01 polypeptide to
polypeptides heterologous thereto, or fragments thereof.
[0133] The lymphocyte obtained from a host animal after
immunization is fused to an immortalized cell line using a fusing
agent such as polyethyleneglycol in order to establish a hybridoma
cell (Goding, 1996). As a cell for fusion, the myeloma cell of a
rodent, cattle, or human, immortalized by transformation or the
myeloma cell line of a rat or mouse is used. After cell fusion,
cells are grown in a suitable culture medium containing one or
pleural substrates inhibiting the growth or survival of non-fused
lymphocyte and immortalized cell line. A typical technique uses a
parent cell lacking the enzyme hypoxanthine guanine
phosphoribosyltransferase (HGPRT or HPRT). In this case,
hypoxanthine, aminopterin, and thymidine inhibit the growth of
HGPRT-deficient cells, and are added to a medium allowing the
growth of hybridoma cells (HAT medium).
[0134] In preparing the monoclonal antibody, a preferable
immortalized cell line is a mouse myeloma strain which is available
from American Type Culture Collection (Manassas, Va.). For the
production of monoclonal antibodies using human myeloma and
mouse-human heteromyeloma cell lines, see Kozbor, et al., 1984.
[0135] Since a hybridoma cell extracellularly secretes an antibody,
whether a monoclonal antibody to the GT01 polypeptide has been
produced or not can be confirmed using a liquid culture medium. The
binding specificity of the monoclonal antibody produced can be
evaluated by an immunoprecipitation technique such as
radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA)
or by in vitro binding assay (Harlow and Lane, 1988; Harlow and
Lane, 1999).
[0136] The anti-GT01 polypeptide monoclonal antibody-secreting
hybridoma cells can be isolated as a single clone by a limiting
dilution method and subculture (Goding, 1996). Suitable culture
media include a Dulbecco's modified Eagle medium, RPMI-1640, and,
in some cases, a protein-free medium or a serum-free medium (for
example, Ultra DOMA PF or HL-1; Biowhittaker; Walkersville, Md.).
In addition, the hybridoma cells may be proliferated in the ascites
fluid of a suitable host animal.
[0137] The monoclonal antibody is isolated and purified from the
medium or ascites fluid using a method well-known to those skilled
in the art, such as protein A sepharose, hydroxyapatite
chromatography, ammonium sulfate precipitation, or affinity
chromatography (Harlow and Lane, 1988; Harlow and Lane, 1999).
[0138] The monoclonal antibody may be also prepared by a gene
recombination technique (U.S. Pat. No. 4,166,452). To identify a
gene encoding a desired monoclonal antibody polypeptide from a
hybridoma cell line secreting an anti-GT01 polypeptide antibody,
for example, oligonucleotide probes specifically binding to mouse
heavy-chain and light-chain antibody genes may be used. As a
result, when the heavy-chain and light-chain antibody genes are
obtained, these genes can be sequenced to identify a desired
antibody gene. In order to express a monoclonal antibody, the DNA
fragment of the identified and isolated antibody gene is introduced
into a suitable expression vector, and the vector is transfected
into a host cell not producing other Ig proteins such as simian
COS-7 cell, Chinese hamster ovary (CHO) cell, or myeloma cell. The
isolated DNA fragment may be modified, for example, by replacing
the coding sequences of the constant domains of the human
heavy-chain and light-chain with homologous mouse sequences (U.S.
Pat. No. 4,816,567; Morrison, et al., 1987), or by fusing part or
all of the sequence encoding a non-Ig polypeptide to the coding
sequence of Ig. The non-Ig polypeptide can be replaced by the
constant domain of antibody or the constant domain of the
antigen-binding site to prepare a chimeric divalent antibody.
[0139] (4)-3. Humanized and Human Antibodies
[0140] The GT01 polypeptide antibody includes a humanized or human
antibody. The humanized form of a non-human antibody is a chimeric
Ig, Ig chain, or fragment (Fv, Fab, Fab', F(ab').sub.2, or the
antigen binding region of a different antibody) containing a
minimal sequence derived from a non-human Ig.
[0141] A humanized antibody generally has one or pleural amino acid
residues introduced from a non-human-derived Ig. These non-human
amino acid residues are often selected from the variable domain.
The humanized antibody can be prepared, for example, by replacing
the CDRs or CDR sequences of a mouse with corresponding human
antibody sequences (Jones, et al., 1986; Riechmann, et al., 1988;
Verhoeyen, et al., 1988). In other words, the humanized antibody
typically means a human antibody in which particular CDR residues
in the human Ig are replaced by CDR residues derived from the
corresponding site of a mouse. The humanized antibody includes a
human Ig substituted by residues having a desired specific affinity
to an antigen, which construct a CDR of a non-human species such as
mouse, rat, or rabbit. In addition, the Fv framework residues of a
human Ig may be substituted by non-human-derived residues (Jones,
et al., 1986; Presta, 1992); Riechmann, et al., 1988).
[0142] (5) A Ligand for the GT01 Polypeptide
[0143] Available methods for determining a ligand include, when the
GT01 receptor protein is allowed to contact with a test compound
(candidate ligand), for example, a method for checking for the
intracellular migration of the receptor protein and a method for
determining the activities of stimulating a cell expressing the
receptor protein, which are operable within the scope of
technological common sense to those skilled in the art.
[0144] Specifically, whether or not a test compound can function as
a ligand (agonist or antagonist) can be examined, for example, by
expressing, on the surface of a test cell, an expression vector for
expressing a chimeric fusion protein between the GT01 receptor
protein according to the invention and a fluorescent protein (e.g.,
GFP, CFP, YFP, or DsRED) and observing, by a fluorescence
microscope or the like, the intracellular migration of the chimeric
protein when the test compound is allowed to contact the expressed
vector. The vector for expressing the chimeric fusion protein with
the fluorescent protein can be constructed by inserting, into a
vector into which a commercial fluorescent protein gene (e.g.,
pDsRed, pEGFP, or pCFP; Clontech) is inserted, the GT01 gene of the
invention so as to be coincident with a frame.
[0145] When a test compound is allowed to contact with a cell on
the surface of which the GT01 receptor protein is expressed, the
cell-stimulating activities (e.g., arachidonic acid release,
acetylcholine release, intracellular Ca.sup.2+ release,
intracellular cAMP formation, inositol phosphate production)
thereof via the receptor protein may be also detected to identify a
candidate ligand. The method for determining the concentration of
free intracellular Ca.sup.2+ is used in the invention. The
concentration of intracellular Ca.sup.2+ can be determined using a
technique well-known to those skilled in the art. By way of
example, a method employing a fluorescent substance emitting
fluorescence by binding to Ca.sup.2+ is generally often used.
[0146] Preferred methods of the invention for identifying a ligand
are a method based on intracellular migration using chimeric fusion
between a fluorescent substance and the GT01 receptor protein and a
method for detecting a change in the concentration of intracellular
Ca.sup.2+.
[0147] 3. Cell Culturing
[0148] To identify a ligand for the GT01 polypeptide of the
invention, the polypeptide needs to be expressed on the surface of
a suitable cell in such a state that it can function.
[0149] The cell which can be used here may be any cell that is a
mammalian cell or an established cell line thereof. A skilled
artisan can easily select a suitable cell or cell line. By way of
example, a CHO, STC-1, GLUTTag, or HEK cell can be used.
[0150] In culturing cells, an MEM medium containing about 5 to 20%
of fetal bovine serum and a DMEM medium maybe, for example, used as
a medium, and, if desired, glucose, glutamine, antibiotic, or the
like is properly added. It is preferred that the pH is about 6 to
8; the temperature, about 37.degree. C.; and the concentration of
CO.sub.2, about 5%.
4. Expression of the GT01 Polypeptide in Mammalian Cells
[0151] To identify the ligand of the invention, it is necessary
that the GT01 polypeptide be expressed in suitable mammalian cells
and be distributed on the surface of the cells.
[0152] To obtain a stable expression strain for the GT01
polypeptide, a DNA encoding the GT01 polypeptide of the invention
(including a substantially identical polypeptide or a partial
peptide thereof; herein after referred to the same) alone or a
fusion polypeptide with another protein (e.g., GFP or G16) is
inserted into a suitable vector (e.g., pCDN), which is then
introduced into desired cells.
[0153] Methods for introducing the vector into animal cells include
a DEAE-dextran method (Lopata, et al., 1984), an electroporation
method, a calcium phosphate method (Chen and Okayama, 1988), and a
method using a cationic lipid (Elroy-Stein and Moss, 1990).
[0154] Available markers for selecting a recombinant cell stably
transformed include, but not limited to, a hygromycin-resistant
marker (Hyg.sup.r), a dihydrofolate reductase gene (dhfr), an
ampicillin-resistant gene (Amp.sup.r), a kanamycin-resistant gene
(Kan.sup.r), and a neomycin-resistant gene (Neo.sup.r, G418).
5. A Pharmaceutical Composition Containing an Agonist or Antagonist
for the GT01 Polypeptide.
[0155] An agonist or antagonist for the GT01 polypeptide can be
used, together with a pharmacologically acceptable vehicle, as a
therapeutic agent in the form of a pharmaceutical composition
having no negative effect on the living body.
[0156] The term "pharmacologically acceptable vehicle" refers to a
vehicle including a solvent, a dispersion medium, a coating agent,
antibacterial and antifungal agents, and an agent retarding
adsorption by isotonic action or a mimetic thereof, and suitable
for pharmaceutical administration (Gennaro, 2000). Examples of the
vehicle and a preferable diluent for the vehicle include, but not
limited to, water, saline, finger solution, dextrose solution, and
5% human serum albumin. A water-insoluble medium such liposome or
fixed oil is also used. In addition, a particular compound which
protects or promotes the activities of the GT01 polypeptide of the
invention and an agonist or antagonist for the polypeptide,
including an anti-GT01 polypeptide antibody can be incorporated
into the composition.
[0157] (1) Preparation of the Pharmaceutical Composition
[0158] The pharmaceutical composition of the invention is
formulated so as to be compatible with therapeutically suitable
administration routes including intravenous or oral administration
and direct administration into the stomach. The solution or
suspension used for intravenous administration or direct
administration into the stomach may contain, but not limited to, a
sterile diluent such as water for injection, saline, fixed oil,
polyethylene glycol, glycerin, propylene glycol, or other synthetic
solvent; a preservative such as benzyl alcohol or other
methylparabens; an antioxidant such as ascorbic acid or sodium
bisulfite; a soothing agent such as benzalkonium chloride or
procaine hydrochloride; a chelating agent such as
ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate,
citrate, or phosphate; and an agent for adjusting osmotic pressure
such as sodium chloride or dextrose.
[0159] The pH may be adjusted using an acid or base such as
hydrochloric acid or sodium hydroxide. A parenteral preparation is
contained in an ample, a glass or plastic disposable syringe, or a
vial for multiple administrations.
[0160] (2) Injectable Preparations
[0161] Pharmaceutical compositions suitable for injection include a
sterilized injectable solution or dispersion medium, wherein it is
a sterile aqueous solution (water-soluble) or dispersion medium for
preparation at the time of use, and a sterilized powder (including
a lyophilized protein or nucleic acid). Suitable vehicles for
intravenous administration include saline, bacteriostatic water,
Cremophor EL.TM. (BASF, Parsippany, N.J.), and phosphate buffered
saline (PBS). When used as an injection, the composition must be
sterile, and must retain sufficient fluidity because it is
administered using a syringe. The composition must be stable e.g.,
to chemical change and corrosion, and must not produce
contamination from microorganisms such as bacteria and fungi during
preparation and storage. The vehicle used here may be a solvent,
including, for example, water, ethanol, polyol (e.g., glycerol,
propylene glycol, or liquid polyethylene glycol), or a suitable
mixture, or a dispersion medium. By way of example, a coating agent
such as lectin is used to maintain a particle size as required for
the dispersion medium, and a surfactant is used to keep moderate
the fluidity. Various antibacterial and antifungal agents such as,
for example, paraben, chlorobutanol, phenol, ascorbic acid, and
thimerosal can be used to prevent the contamination of
microorganisms. An agent keeping isotonicity such as polyalcohol
(e.g., sugar, mannitol, or sorbitol) or sodium chloride may be
contained in the composition. The composition capable of retarding
adsorption contains an agent such as aluminum monostearate or
gelatin.
[0162] The sterile injectable solution is prepared by adding
necessary components alone or a combination thereof with other
components to a suitable solvent, to which a necessary amount of an
active compound is then added before sterilization. The dispersion
medium is typically prepared by incorporating an active compound
into a sterile medium containing a basic dispersion medium and the
above-described other necessary components. Methods for preparing a
sterile powder for preparing a sterile injectable solution include
vacuum drying and freeze drying for preparing a powder containing
an active ingredient and any desired component derived from the
sterile solution.
[0163] (3) Oral Compositions
[0164] The oral composition typically contains an inactive diluent,
or a vehicle which is harmless even when incorporated into the
body. The oral composition is, for example, included in a capsule
of gelatin, or pressurized for tabletation. For oral treatment, an
active compound is incorporated together with an excipient, and
used in the form of a tablet, troche, or capsule. The oral
composition can be also formulated using a flowable vehicle, and
the composition in the flowable vehicle is orally applied. In
addition, a pharmaceutically suitable binder and/or an adjuvant
substance or the like maybe included.
[0165] The tablet, pill, capsule, troche, and the like can contain
the following components, or compounds having similar properties:
an excipient such as microcrystalline cellulose; a binder such as
gum arabic, tragacanth, or gelatin; a swelling agent such as
starch, lactose, alginic acid, Primogel, or corn starch; a
lubricant such as magnesium stearate or Strrotes; a lubricating
agent such as colloidal silicon dioxide; a sweetening agent such as
sucrose or saccharin; and a flavoring agent such as peppermint,
methylsalicylic acid, or orange flavor.
[0166] (5) Systemic Administration
[0167] The systemic administration may be carried out
transmucosally or percutaneously. For the transmucosal or
percutaneous administration, a penetrant capable of permeating a
target barrier is selected. Transmucosal penetrants include
surfactants, bile salts, and fusidic acid derivatives. A transnasal
spray or a suppository may be used for the transmucosal
administration. For the transmucosal administration, an active
compound is formulated in the form of an ointment, salve, gel, or
cream.
[0168] For delivery to the rectum, a compound may be also
formulated in the form of a suppository (together with, for
example, cocoa butter and another base such as glyceride) or a
retentive enema.
[0169] (6) Vehicles
[0170] The GT01 polypeptide of the invention and an agonist or
antagonist for the polypeptide, including an anti-GT01 polypeptide
antibody may be formulated in the form of a controlled release
preparation such as an implant or a microencapsulated delivery
system, using a vehicle capable of preventing the immediate removal
thereof from the body. Biodegradable, biocompatibile polymers such
as ethylene vinyl acetate, polyethylene glycol, polyacid anhydride,
polyglycolic acid, collagen, polyorthoester, and polylactic acid
may be used. These materials can be obtained from ALZA Corporation
(Mountain View, Calif.) or NOVA Phamaceuticals, Inc. (Lake
Elsinore, Calif.), and also can be readily prepared by a skilled
artisan. A suspension of liposome may be also used as a
pharmaceutically acceptable vehicle. By way of non-limiting
example, a useful liposome is prepared as a lipid composition
containing phosphatidylcholine, cholesterol, and PEGylated
phosphatidylethanol (PEG-PE) by passing through a filter having a
suitable pore size so as to provide a size suited for use, and
purified using a reverse-phase evaporation method. By way of
example, the Fab' fragment of antibody or the like maybe also bound
to the liposome via disulfide exchange reaction (Martin and
Papahadjopoulos, 1982). For details regarding methods for the
preparation, see, for example, descriptions in Eppstein, et al.,
1985; Hwang, et al., 1980.
[0171] (7) Dosages
[0172] In the treatment or prevention of a particular disease using
the GT01 polypeptide of the invention, a gene encoding the
polypeptide, or the like, suitable dosage levels thereof depend on
the condition of a patient to be treated, the method of
administration, and the like, but can be easily optimized by one of
skill in the art.
[0173] For administration by injection, for example, a daily dose
of about 0.1 .mu.g to 500 mg per kg of body weight of a patient is
preferably administered, and may be generally given at a time or in
divided portions. The dosage level is preferably about 0.1 .mu.g/kg
to about 250 mg/kg per day, more preferably about 0.5 to about 100
mg/kg.
[0174] For oral administration, the composition is preferably
provided in the form of a tablet containing 1.0 to 1,000 mg of
active ingredient, and the amount of the effective active
ingredient present in a dose for a patient to be treated is
preferably 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0,
150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0,
900.0, or 1,000.0 mg. The compound is administered with a dosage
regimen of once to four times a day, preferably once to twice a
day.
[0175] (8) Unit Dosage
[0176] The pharmaceutical composition or preparation must comprise
a uniform unit dosage to secure a constant dosage. The unit dosage
means a unit containing a single dose effective for treating a
patient and formulated together with a pharmaceutically acceptable
vehicle. The determination of the unit dosage of the invention is
influenced, for example, by the physical and chemical
characteristics of a compound (e.g., free fatty acid or anti-GT01
polypeptide antibody) to be formulated, the expected therapeutic
effect thereof, and points of concern in formulation typical for
the compound.
6. Gene Therapy Composition
[0177] To introduce the nucleic acid molecule (for example, a
vector into which a polynucleotide encoding the GT01 polypeptide is
inserted) disclosed in the invention into cells of a patient, there
are two major methods, in vivo and ex vivo. For in vivo delivery,
it is directly injected into the site of a patient required to be
treated. For ex vivo treatment, cells at the site of a patient
required to be treated can be isolated, into which a formulated
nucleic acid molecule is then introduced, followed by administering
the resultant cells directly or, for example, after encapsulation
in a porous membrane to be embedded in the patient (see U.S. Pat.
Nos. 4,892,538 and 5,283,187). A technique available for
introducing the nucleic acid molecule into live cells is selected
depending on whether it is introduced into cultured cells or the
like in vitro or into a patient in vivo. Techniques suitable for
introducing the nucleic acid molecule into mammalian cells in vitro
include liposome, electroporation, microinjection, transfection,
cell fusion, DEAE-dextran method, and calcium phosphate method. The
transfection comprises the binding of the particle of a recombinant
virus (preferably, retrovirus) to a cellular receptor and the
subsequent introduction of the nucleic acid molecule contained in
the particle into the cell. The vector typically used for the ex
vivo delivery of a gene is retrovirus.
[0178] Current preferred techniques for in vivo nucleic acid
transfer include systems using a viral or non-viral vector
(adenovirus, lentivirus, Herpes simplex I virus, or
adeno-associated virus (AAV)) and a cationic lipid-based system (a
lipid useful for the lipid-mediated transfer of a gene is, for
example, DOTMA, DOPE, or DC-Chol; see, for example, Tonkinson, et
al., Cancer Investigation, 14 (1): 54-65 (1996)). The most
preferable vectors for use in gene therapy are viruses; among
others, adenovirus, AAV, lentivirus, or retrovirus is most
preferable. Viral vectors such as a retroviral vector contain at
least one transcriptional promoter/enhancer or positioning factor.
In addition, for example, in the case of transcription in a state
containing a gene encoding the G01 polypeptide, viral vectors such
as a retroviral vector contain a cis element enabling the
translation of the coding gene, that is, a nucleic acid sequence
functioning as a translation initiation sequence. These vector
constructs contain a packaging signal or long terminal repeat (LTR)
or portion thereof suitable for the virus used. In some cases, the
vector constructs also contain polyadenylation and translational
termination sequences. For example, they contain a 5' LTR,
tRNA-binding site, a packaging signal, a DNA synthesis initiation
point, and a 3' LTR or portion thereof. Other non-viral vectors
maybe, for example, cationic lipids, polylysines, or
dendrimers.
[0179] In some cases, the vector is preferably provided together
with an agent targeting a nucleic acid used for treatment at
desired cells, such as, for example, an antibody specific for a
cell-surface membrane protein. For review of the currently known
gene labeling and gene therapy protocols, see Anderson, et al.,
Science, 256: 808-813 (1992). For suitable gene therapy and methods
for making retroviral particles and structural proteins, see U.S.
Pat. No. 5,681,746.
7. A Kit for the Pharmaceutical Composition
[0180] The pharmaceutical composition may be included together with
an explanation for administration in a kit, container, or pack.
When the pharmaceutical composition according to the invention is
supplied in the form of a kit, the different constituents of the
pharmaceutical composition are packaged in separate containers, and
mixed immediately before use. The separate packaging of
constituents is intended to enable the long-term storage thereof
without the loss of function of an active constituent.
[0181] (1) Containers
[0182] Reagents contained in the kit are provided in such a kinds
of container that the constituents effectively maintain the
activities thereof for a long period of time, are not adsorbed to
the material of the container, and cause no deterioration. By way
of example, a sealed glass ample contains a buffer packaged under a
neutral and unresponsive gas such as nitrogen. An ample is composed
of glass, an organic polymer such as polycarbonate or polystyrene,
ceramic, metal, or any of other suitable materials typically used
for holding reagents. Other suitable containers include, for
example, a simple bottle made of a material similar to that of an
ample or the like, and a packaging material whose inside is lined
with a foil such as aluminium or alloy. Other containers include a
test tube, a vial, a flask, a bottle, a syringe, and a container
similar thereto. Containers have a sterile access port, such as a
bottle having a stopper which can be pierced by a hypodermic
injection needle.
[0183] (2) Instructions
[0184] The kit also includes instructions. Instructions for the use
of the kit comprising the pharmaceutical composition may be printed
on paper or other materials, or supplied in the form of an
electrically or electromagnetically readable medium such as
floppy.TM. disk, CD-ROM, DVD-ROM, Zip disk, video tape, or audio
tape. Detailed instructions may be actually included in the kit, or
may be carried on a website designated or informed through an
electronic mail system or the like by the manufacturer or
distributor of the kit.
8. A Dietary Supplement Composition or Dietary Supplement Food
Containing an Agonist or Antagonist for the GT01 Polypeptide.
[0185] When an agonist or antagonist for the GT01 polypeptide is
used to make the dietary supplement composition or dietary
supplement food according to the invention, generally, the shape of
the food is not particularly restricted, but is preferably that
enabling the long-term ingestion thereof as an ordinary food;
examples thereof can include a tablet, a granule, a powder, a
refreshing drink, confectionary, bread, and margarine. In addition,
an additive, extender, perfume, sweetener, thickener or the like
usually used in foods may be properly mixed as far as advantages of
the invention are not impaired.
9. Other Medical and Pharmaceutical Applications of the GT01
Protein or a Partial Peptide Thereof
[0186] The GT01 polypeptide of the invention or a partial peptide
thereof, or a salt thereof and polynucleotides encoding them can be
used, for example, for: (i) the determination of a ligand (agonist)
for the GT01 protein; (ii) an agent for preventing and/or treating
diseases associated with the dysfunction of the GT01 protein; (iii)
an agent for gene diagnosis; (iv) the quantitative determination of
the ligand for the GT01 protein; (v) the screening of a compound
altering the binding properties of the ligand to the GT01 protein;
(vi) an agent for preventing and/or treating various diseases,
containing the compound altering the binding properties of the
ligand to the GT01 protein; (vii) the quantitative determination of
the GT01 protein or a partial peptide thereof; (viii) the
neutralization of the GT01 protein or a partial peptide thereof by
an antibody thereto; and (ix) the preparation of a non-human animal
having a gene encoding the GT01 protein. Particularly, a receptor
binding assay system employing an expression system for the
recombinant GT01 protein of the invention may be used to screen a
substance (for example, agonist or antagonist) altering the binding
properties of a human- or mammal-specific GT01 protein to a ligand
therefor; the resultant agonist or antagonist can be used, for
example, as an agent for preventing or treating various
diseases.
[0187] Specifically, the GT01 protein of the invention or a partial
peptide of the same, or a salt thereof is useful as a reagent for
screening or identifying a ligand (agonist) or antagonist for the
GT01 protein. These reagents can each provide a component of a kit
for screening a substance altering the binding properties of the
ligand to the GT01 protein. As described above, the present
invention provides a method for determining a ligand (agonist) or
antagonist for the GT01 protein, characterized in that the GT01
protein or a partial peptide of the same, or a salt thereof is
allowed to contact with a test substance. Examples of the test
substance include human or mammal (e.g., mouse, rat, pig, cattle,
sheep, or monkey) tissue extracts, cell culture supernatants, and
artificially synthesized compounds.
[0188] To perform a method for determining a ligand for the GT01
protein of the invention or a salt thereof, an appropriate GT01
protein fraction and a labeled test substance are used. Preferably,
the GT01 protein fraction is, for example, a natural GT01 protein
fraction or a recombinant GT01 protein fraction having an activity
comparable to that thereof. To determine a ligand for the GT01
protein or a salt thereof, for example, cells or membrane fractions
thereof containing the GT01 protein are first suspended in a buffer
suitable for the determination method to prepare a GT01
preparation. The buffer may be any buffer which does not inhibit
the binding of a ligand to a receptor protein, including a
phosphate buffer or a Tris-hydrochloric acid buffer. For the
purpose of reducing non-specific binding, a surfactant such as
CHAPS, Tween-80, or deoxycholate, or a protein such as bovine serum
albumin or gelatin may be also added to the buffer. A given amount
of radiolabeled test substance is allowed to coexist in a solution
containing the GT01 protein. For the purpose of knowing the amount
of non-specific binding, a reaction tube to which a large excess of
the unlabeled test compound has been added is also provided. The
reaction is conducted at about 4 to 50.degree. C., preferably about
4 to 37.degree. C. for about 10 minutes to 24 hours, preferably
about 30 minutes to 3 hours. After the reaction, filtration and
subsequent washing with an appropriate amount of the same buffer
are carried out, followed by measuring the remaining radioactivity
on the filter paper using a liquid scintillation counter or the
like. A test substance for which the count corresponding to the
total amount of binding, less the amount of non-specific binding
exceeds 0 cpm can be selected as a ligand (agonist) for the GT01
protein of the invention or a salt thereof.
[0189] The following Examples are provided for illustrative
purposes only, and not intended to limit the scope of the invention
in any manner.
[0190] All patent documents and references cited in this
specification are incorporated herein by reference in their
entirety.
EXAMPLE 1
Cloning of a GT01 Gene
[0191] cDNA was prepared by subjecting 5 .mu.g of total ileal RNA
in a human organ RNA panel or total RNA extracted from mouse ileum
to reverse transcription using Random primer (from Takara)
according to a method included with SuperScipt II (from
Invitrogen). After the reaction, a 5' primer
(5'-ATGTCCCCTGAATGCGCGCGGG-3') (SEQ ID NO: 3) and a 3' primer
(5'-GCCAGAAATAATCGACAAGTCA-3') (SEQ ID NO: 4) were employed to
perform RT-PCR using TaKaRa EX Taq (from Takara). PCR reaction was
carried out by denaturing the cDNA at 95.degree. C. for 2 minutes
and then conducting 35 reaction cycles of 96.degree. C. for 30
seconds, 52.degree. C. for 30 seconds, and 72.degree. C. for 2
minutes to amplify the PCR product. Then, the resultant product was
subjected to elongation reaction at 72.degree. C. for 5 minutes,
and the reaction was terminated by cooling to 4.degree. C. The PCR
fragment was subcloned into pGEM-T easy (from Promega) vector and
then sequenced. Each of the human and mouse fragments was cut out
with a restriction enzyme, and the whole thereof was placed
downstream of the promoter of an expression vector, pIRES (from
Clonetech) so as to express the full length. Further, in conducting
PCR, primers from which stop codons were removed were prepared, and
a full-length cDNA was produced in the same procedure. The
resultant cDNA was introduced into a pEGFP-N3 expression (from
Clonetech) vector or the expression vector whose EGFP sequence was
replaced by G16 to make an expression vector capable of producing a
fusion protein with EGFP or G16.
EXAMPLE 2
Tissue Distribution of Gene Expression
[0192] (1) Preparation of Tissues
[0193] C57BL/6 male mice were anaesthetized with ether, and
subjected to perfusion fixation using 4% paraformaldehyde/0.1M
phosphate buffer (pH 7.4). Then, part of the colon was taken, the
content thereof was removed in cooled phosphate-buffered saline
(PBS), and fixation was then carried out at 4.degree. C. for one
day. Subsequently, replacement was performed with 20% sucrose/0.1M
phosphate buffer (pH 7.4) at 4.degree. C. for two days or more. The
sample subjected to the replacement was treated with O.C.T Compound
and frozen using liquid nitrogen and stored at -80.degree. C. until
use. A fresh frozen sample was prepared as described below. Male
mice of the same strain were anaesthetized with ether, part of the
jejunum or colon was taken, and the intestinal content was then
washed with cooled PBS. The water was slightly drained from the
sample, which was then rapidly embedded using O.C.T Compound,
followed by freezing with liquid nitrogen and storing at
-80.degree. C. until use.
[0194] (2) RT-PCR
[0195] Total RNA was extracted from each eviscerated mouse organ
using ISOGEN (Nippon Gene). The resultant total RNA (5 mg) was
subjected to RT reaction using Ready-To-Go You Prime First-Strand
Beads(from Amersham Bioscience, Sweden) to prepare cDNA. After the
reaction, a 5' primer (5'-CGCACCCGCTTTCCCTTCTTCTC-3') (SEQ ID NO:
3) and a 3' primer (5'-AGCTCT TTCCTTGATGCCTTTGTGA-3') (SEQ ID NO:
4) were employed to perform RT-PCR using TaKaRa EX Taq (from
Takara). PCR reaction was carried out by denaturing cDNA at
95.degree. C. for 2 minutes and then conducting 35 reaction cycles
of 96.degree. C. for 30 seconds, 52.3.degree. C. for 30 seconds,
and 72.degree. C. for 2 minutes to amplify the PCR product. Then,
the resultant product was subjected to elongation reaction at
72.degree. C. for 5 minutes, and the reaction was terminated by
cooling to 4.degree. C.
[0196] (3) Southern Hybridization
[0197] After the RT-PCR reaction, the products were electrophoresed
using 2% agarose gel, and transcribed to a nitrocellulose membrane.
To prepare a probe for southern hybridization, the sequence of a
mouse GT01 gene (SEQ ID NO: 5) cloned in pGEM-T Easy Vector (from
Takara) was first cleaved with each of the restriction enzymes
BssHI and BglII, and subjected to electrophoresis using 1% agarose
gel. After cutting out a desired DNA band, the DNA was purified
using Geneclean II (from Q-BIO gen, USA) to make a template for the
probe. A mouse GT01 gene-specific DNA probe was prepared by Random
Primer DNA labeling Kit Ver. 2 (from Takara) using .sup.32P-labeled
dCTP (from NEN, USA). The .sup.32P-labeled DNA probe was added to
the Southern blotted membrane in a hybridization buffer
(5.times.SSC, 5.times. Denhart's solution, 0.5% SDS), followed by
reaction overnight at 55.degree. C. After the hybridization, the
probe was washed, at 55.degree. C. for 10 minutes, with
2.times.SSC/0.1% SDS and then 0.2.times.SSC. The membrane was
exposed to Fuji Imaging Plate (from Fuji Photo Film), and scanned
using an image analyzer (Storm 860, Amersham Bioscience,
Sweden).
[0198] (4) Results
[0199] The results of the tissue distribution of GT01 gene
expression are shown in FIG. 1. The expression was observed
frequently in the caecum and large intestine, relatively often in
the brain and lung, and also in the rectum, pancreas, and islet
cells. In addition, it was demonstrated that the gene was often
expressed also in the enteroendocrine cell line STC-1. In contrast,
the expression in the heart, liver, and kidney was less often
observed (FIG. 2).
EXAMPLE 3
CCK Immunohistochemistry (see Japanese Patent Application No.
2003-180375 Specification)
[0200] A fresh frozen section of mouse jejunum was sliced into a
thickness of 8 mm using a cryostat (LEICA CM1800; Leica), attached
to an APS-coated slide glass (Matsunami Glass), and air-dried at
-20.degree. C. Then, the slice was fixed in Zamboni solution for 30
minutes before washing with flowing water for 10 minutes. To block
endogenous peroxidase, treatment with 0.5% sodium metaperiodate was
carried out for 10 minutes before washing with flowing water for 10
minutes. The non-specific reaction of an anti-CCK antibody was
blocked using an antibody dilution solution (1% normal bovine
serum, 0.4% Triton-X 100, PBS dilution) forone hour,
followedbywashingwith PBS. The slide glass was transferred to a wet
box, and allowed to react with a rabbit anti-CCK antibody (1:4,000,
AB1972, Chemicon, USA) overnight at room temperature. After the
reaction, it was subjected thrice to a 5 minutes of washing with
PBS, allowed to react with a biotin-labeled goat anti-rabbit IgG
(1:2,000, Cat. No. 55701, ICN Pharmaceuticals, USA) at room
temperature for 2 hours, and then subjected thrice to a 5 minutes
of washing with PBS. Subsequently, an avidin-biotin-peroxidase
complex (Vectastain ABC Kit, Vector Labs, USA) was allowed to react
therewith for 40 minutes, followed by subjecting thrice to a 5
minutes of washing with PBS. Then, it was color-developed with a
DAB reaction solution (a 50 mM Tris buffer (pH 7.6) containing
0.02% 3,3-diaminobenzidine tetrahydrochloride and 0.06% hydrogen
peroxide solution). After the color development, it was washed with
flowing water and subjected to dehydration and penetration using an
ethanol-xylene series, and the sample was then mounted using a
mounting agent (MP500, Matsunami Glass).
EXAMPLE 4
In Situ Hybridization
[0201] (1) Preparation of a cRNA Probe
[0202] The mouse GT01 sequence cloned in pGEM-T Easy Vector
(Takara) was cleaved with a restriction enzyme, SpeI, for preparing
a sense probe or with a restriction enzyme, NcoI, for preparing an
anti-sense probe. The resultant linear plasmid DNAs were each used
in an amount of 1 mg for cRNA probe synthesis; using DIG RNA
Labeling Kit (Roche Diagnostics, Switzerland), the total amount of
the reaction mixture (plasmid DNA, 1.times. DIG RNA labeling Mix,
1.times. Transcription buffer, 1 U/ml RNasin, 2 U/ml T7 or SP6RNA
polymerase, RNase-free dH.sub.2O) was set to 20 ml. This reaction
solution was allowed to react at 37.degree. C. for 2 hours,
followed by decomposing the plasmid DNA using a DNAase before
terminating the reaction employing 1 ml of 0.5M EDTA. The
synthesized cRNA probe was ethanol precipitated, and the pellet
obtained by centrifugation (15,000 rpm, 4.degree. C., 15 minutes)
was dried, which was then dissolved in an alkaline hydrolyzing
liquid (40 mM NaHCO.sub.3, 60 mM Na.sub.2CO.sub.3, pH 10.2) before
subjecting to fragmentation treatment at 60.degree. C. for 9
minutes. After the treatment, ethanol precipitation was again
carried out, and the precipitate was dissolved in DEPC water
(Milli-Q water treated with 0.1% DEPC overnight and then heated in
an autoclave at 121.degree. C. for 40 minutes for
detoxication).
[0203] (2) In Situ Hybridization
[0204] The frozen sample of mouse colon fixed with 4% paraform
aldehyde was sectioned in to a thickness of 20 mm using a cryostat
(Leica CM1800; Leica) and floated on 4.times.SSC (0.6 M NaCl, 0.6 M
sodium citrate). The resultant section was washed with PBS and
treated with 1 mg/ml Proteinase K (dilution with 0.1 M Tris-HCl (pH
8.0)/50 mM EDTA) at 37.degree. C. for 20 minutes. It was fixed with
4% paraform aldehyde for 10 minutes and then washed with PBS. This
was allowed to stand in acetic anhydride (dilution with 0.1 M
triethanolamine) at room temperature for 10 minutes, and again
washed with PBS. Then, the probe was added to a hybridization
buffer (50% formamide, 10 mM Tris-HCl (pH 7.6), 1.times. Denhardt
Solution, 0.2 mg/ml yeast tRNA, 10% dextran sulfate, 600 mM NaCl,
0.25% SDS, 0.5 M EDTA (pH 8.0)) so as to provide a concentration of
200 ng/ml, which was allowed to react at 60.degree. C. overnight
(for about 16 hours). After the hybridization reaction, the probe
was washed with 2.times.SSC/50% formamide at 60.degree. C. for 30
minutes, which was then replaced by TNE (10 mM Tris-HCl (pH 7.6),
500 mM NaCl, 1 mM EDTA) for 10 minutes, and excess probes were
digested using a 20 mg/ml RNase (dilution with TNE). Washing was
carried out with TNE for 10 minutes, followed by washing with
2.times.SSC, 1.times.SSC and 0.5.times.SSC at 55.degree. C. for 20
minutes. To detect a signal, replacement by TBS (100 mM Tris-HCl
(pH 7.5), 150 mM NaCl) was performed for 5 minutes, followed by the
reaction of blocking a DIG antibody at 37.degree. C. for one hour
using a 1.5% blocking reagant (dilution with TBS). Washing was
carried out using TBS for 5 minutes, and antibody reaction was
performed at room temperature for one hour using a goat ant-DIG
antibody (Roche Diagnostics, Switzerland) 1:500 (dilution with 1.5%
blocking reagent)). The antibody was removed by washing with TBST
(100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20), and
replacement by NTM (100 mM Tris-HCl (pH9.5), 100 mM NaCl, 50 mM
MgCl.sub.2) was carried out for 3 minutes. Then, color development
was performed under microscopic examination using 0.34 mg/ml NBT
and 0.18% BCIP (dilution with NTM), and the color development
reaction was terminated by treatment with a reaction termination
solution (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) for 10 minutes. After
the color development, the section was placed on a slide glass in
PBS, mounted with 90% glycerol (dilution with PBS), and subjected
to microscopic examination using an optical microscope.
EXAMPLE 5
Preparation of Stable Expression Cells
[0205] To obtain a vector containing a desired gene, EGFP in
pEGFP-N3 (from Invitrogen) was cut out using restriction enzymes,
KpnI and NotI (from Takara), and the DNA sequence of G16 was
inserted employing TaKaRa Ligation Kit ver. 2 (from Takara). In
addition, the sequence of mouse GT01 was inserted upstream of G16
using the restriction enzyme, KpnI (from Takara) and TaKaRa
Ligation Kit ver. 2 (from Takara).
[0206] The transfection of the DNA into cells was carried out using
an electroporation method. Cells (HEK-293 (derived from human fetal
kidney, 2,500,000 cells) were suspended in a medium (Dulbecco's
Modified Eagle Medium, high glucose, GIBCO), and the DNA solution
(DNA amount: 10 to 15 .mu.g) was then added. After 10 minutes, DNAs
were transfected into cells under the conditions of 240 V and
975-.mu.F using Bio Rad Capacitance Pulse Controller Gene
Pulser.
[0207] The cells transfected with the receptor DNA were cultured in
an agent-containing medium (G418: 1.0 mg/mL, penicillin: 100 units
/mL, streptomycin: 100 .mu.g/mL, 10% FCS) at 37.degree. C. and 5%
CO.sub.2 for selection. After 10 days, colonies were picked up and
cultured in an agent-containing medium (G418: 0.5 mg/mL,
penicillin: 100 units /mL, streptomycin: 100 .mu.g/mL, 10% FCS)
EXAMPLE 6
Measurement using the Intracellular Migration of the Receptor (GT01
Polypeptide)
[0208] (1) E-C-L Coating of a Plate for Assay
[0209] Sterile PBS (137 mM NaCl, 8.1 mM
Na.sub.2HPO.sub.4.12H.sub.2O, 2.68 mM KCl, 1.47 mM
KH.sub.2PO.sub.4) containing 5 .mu.g of E-C-L Cell Attachment
Matrix (from Upstate) was added to a View Plate-96 (from Packard)
so as to provide an amount of 100 .mu.L/ well, and cultured at
37.degree. C. for one hour or at 4.degree. C. overnight. This was
used for the following assay.
[0210] (2) Seeding Cells on the Plate
[0211] Cells (HEK cells) stably expressing a chimeric receptor were
detached with trypsin and suspended in a medium containing 10% FCS.
The cells were seeded on the E-C-L-coated plate so as to provide a
liquid amount of 100 .mu.L/well and a cell number of
5.times.10.sup.4/well, and cultured overnight under conditions of
37.degree. C. and 5% CO.sub.2, followed by removing the medium and
adding a serum-free medium in an amount of 100 .mu.L/well.
[0212] (3) Assay of an Agonist (or Antagonist)
[0213] A lipid assumed to be an agonist (or antagonist) for the
chimeric receptor expressed on the cell was added so as to provide
an amount of 1 .mu.L/well. Cells were maintained under conditions
of 37.degree. C. and 5% CO.sub.2 for one hour.
[0214] Fixation and Staining of Cells
[0215] After culturing, the medium was removed, and a fixing and
staining solution (containing 10 .mu.g/mL Hoechst No. 33342 (from
Sigma) and 2% paraformaldehyde (from Nakarai)) was added in an
amount of 100 .mu.L/well, followed by allowing to stand for 30
minutes in the dark.
[0216] Elisa Tape (from Iwaki) was affixed to the plate so as to
completely cover the wells.
[0217] (4) Assay
[0218] The ArrayScan System from Cellomics, Ltd. was used for
analysis. The nucleus was stained with Hoechst and the behavior of
the receptor associated with agent treatment was tracked as the
behavior of a GFP-receptor chimera protein. It is known that some
G-protein coupled receptors localized on cell membrane are
internalized into cytoplasm by ligand stimulation. The chimeric
receptors located at fixed distances from the nucleus were
determined as having been internalized, and the proportion of cells
in which the receptor was internalized to the total number of cells
was calculated for each well. Based on the calculated value, it was
determined whether the lipid used was an agonist (or antagonist)
for the mouse GT01 receptor (FIG. 3).
EXAMPLE 7
Measurement of Intracellular Ca.sup.2+ Concentration
[0219] (1) Measurement using FLIPR
[0220] The concentration of intracellular calcium was measured as
described below. Cells (HEK cells; 200,000cells per well) on which
a desired receptor was stably expressed were cultured on a 96-well
plate (Collagen Cell ware 96-well Black/Clear Plate, Becton
Dickinson) for 20 hours in conditions of 37.degree. C. and 5%
CO.sub.2. FLIPR Calcium Assay Kit (from Molecular Devices) diluted
with a buffer (HEPES/Hanks, pH7.4) was added, followed by
incubating at 37.degree. C. and 5% CO.sub.2 for one hour. Diluted
with the same buffer, a test agent (a free fatty acid such as
capric acid, lauric acid, myristic acid, pentadecanoic acid,
octanoic acid, palmitic acid, stearic acid, arachic acid, behenic
acid, margaric acid, palmitoleic acid, eicosatrienoic acid, elaidic
acid, petroselinic acid, oleic acid, .alpha.-linolenic acid,
.gamma.-linolenic acid, homo-.gamma.-linolenic acid, arachidonic
acid, eicosadienoic acid, eicosatrienoic acid, eicosapentaenoic
acid, docosahexaenoic acid, linoleic acid, eicosatetraenoic acid,
or vaccenic acid) was added, followed by measuring fluorescence
intensity at 510 to 570 nm for excitation light of 488 nm using
FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices) (FIG.
4). FIG. 5 shows pEC.sub.50s for the elevation of Ca.sup.2+
concentration in an HEK cell when various free fatty acids were
added. The fatty acids shown in the data of FIG. 5 are myristic
acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0),
palmitoleic acid (C16:1), margaric acid (C17:0), stearic acid
(C18:0), oleic acid (C18:1), .alpha.-linolenic acid (C18:3),
eicosadienoic acid (C20:2), eicosatrienoic acid (C20:3), and
eicosatetraenoic acid (C20:4). Eicosapentaenoic acid,
docosahexaenoic acid, or other free fatty acids also had comparable
pEC.sub.50.
[0221] (2) Measurement using CAF
[0222] Cells (2,500,000 cells) on which a desired receptor was
stably expressed were suspended in 5 mL of buffer (135 mM NaCl, 5
mM KCl, 10 mM glucose, 10 mM HEPES, 1.2 mM CaCl.sub.2, 1 mM
MgCl.sub.2), to which 15 .mu.L of fura2-AM was then added, followed
by penetration culture at 37.degree. C. for 40 minutes. Then, a
test agent was added thereto, followed by measuring the ratio of
fluorescence intensities at 500 nm for two excitation waves of 340
nm and 380 nm using CAF-110 (Jasco).
[0223] (2)-1. Microscopic Measurement
[0224] Cells (STC-1 cells) are cultured in a 35-mm culture dish
with a cover glass in the bottom. After washing with a Ca-tyrode
solution, a Ca-tyrode solution containing 2 .mu.M fura-AM is added
thereto, which is then placed at room temperature for 20 minutes.
After washing twice with a Ca-tyrode solution, 1 mL of Ca-tyrode
solution is added thereto, and images are captured at room
temperature at 15-second intervals using ARGUS200 (340/380 nm
measurement) with a 40.times. objective lens. For each of the
captured images, the ratio is calculated. Stimulation (using
bombesin) serving as a control for the ligand is carried out 10 and
20 minutes later.
[0225] When an RNAi vector was transfected, fluorescence due to GFP
simultaneously transfected is measured; a Ca.sup.2+ response over
time for cells having GFP fluorescence and showing Ca.sup.2+
elevation with the control vector was quantitatively determined,
and is indicated (FIG. 6). The fatty acid used here is
.alpha.-linolenic acid (C18:3).
[0226] (2)-2. Preparation and Transfection of an RNAi Vector
[0227] A pSilencer 2.1-U6 system from Ambion was used. According to
a instruction included therewith, the oligo pair selected from a
desired gene was synthesized, annealed, and then ligated into the
above-described vector. The resultant construct was sequenced for
confirmation.
[0228] The gene transfection into STC-1 cells was carried out using
Lipofectamine Plus, and intracellular Ca.sup.2+ concentration was
then measured under a microscope (lower graph in FIG. 6). As a
result, the RNAi specific for GT01 eliminated the peak (upper graph
in FIG. 6) of elevated calcium concentration observed by the
addition of .alpha.-linolenic acid (lower graph in FIG. 6). Thus,
it has been demonstrated that .alpha.-linolenic acid elevates
intracellular calcium concentration via GT01.
EXAMPLE 8
Measurement of CCK (See Japanese Patent Application No. 2003-180375
Specification)
[0229] ST-1 cells were cultured in a 24-well plate at densities of
8.times.10.sup.4 and 1.times.10.sup.5 cells/cm.sup.2.
Cholecystokinin octapeptide (26-33,
Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2) was quantitatively determined
24 to 48 hours later. The cells were washed thrice with Hank's
buffer (HBBS), and then reacted with various concentrations of each
free fatty acid in 0.5 mL of Hank's buffer at 37.degree. C. for 60
minutes. The culture supernatant was recovered, centrifuged (at
about 5, 000 g) for 5 minutes to remove cell fragments, and
subjected to the measurement of CCK with a kit using the EIA method
specific for CCK (26-33) (Phoenix Pharmaceuticas Inc., Belmont,
Calif.) (FIG. 7). Here, linolenic acid (C18:3), oleic acid (C18:1),
stearic acid (C18:0), and pelargonic acid (C9:0) were used.
EXAMPLE 9
Measurement of GLP-1
[0230] STC-1 cells were cultured in a 24-well plate at densities of
8.times.10.sup.4 and 1.times.10.sup.5 cells/cm.sup.2. GLP-1 was
quantitatively determined 24 to 48 hours later. The cells were
washed thrice with Hank's buffer (HBBS), and then reacted with
various concentrations (10, 30, and 100 .mu.M) of each free fatty
acid in 0.5 mL of Hank's buffer at 37.degree. C. for 60 minutes.
The culture supernatant was recovered, centrifuged (at about 5,000
g) for 5 minutes to remove cell fragments, and subjected to the
measurement of GLP-1 with a kit using the EIA method specific for
GLP-1 (Phoenix Pharmaceuticas Inc., Belmont, Calif.) (FIG. 8).
Here, .alpha.-linolenic acid, docosahexaenoic acid, palmitoleic
acid, oleic acid, stearic acid, octanoic acid, and
.alpha.-linolenic acid-methyl ester were used (FIG. 8).
EXAMPLE 10
In Situ Hybridization
[0231] (1) Preparation of cRNA Probes
[0232] Labeled probes were prepared from the sequences of mouse and
human GT01 and mouse and human GLP-1 by an in vitro transcription
method. The labeling with digoxigenin was carried out using DIG RNA
Labeling Kit (Roche Diagnostics, Switzerland) and setting the total
amount of a reaction mixture (plasmid DNA, 1.times. DIG RNA
labeling Mix, 1.times. Transcription buffer, 1 U/ml RNasin, 2 U/ml
T7 or SP6 RNA polymerase, RNase-free dH.sub.2O) to 20 ml. This
reaction solution was subjected to reaction 37.degree. C. for 2
hours before digesting the plasmid DNA using a DNase, followed by
terminating the reaction employing 1 ml of 0.5 M EDTA. The
synthesized cRNA probes were each ethanol precipitated and
centrifuged (at 15,000 rpm and 4.degree. C. for 15 minutes) before
drying the resultant pellet, followed by dissolution in an alkaline
hydrolyzing solution(40 mM NaHCO.sub.3, 60 mM Na.sub.2CO.sub.3, pH
10.2) before fragmentation treatment at 60.degree. C. for 9
minutes. After the treatment, ethanol precipitation was again
performed, and the precipitate was dissolved in DEPC water (Milli-Q
water treated with 0.1% DEPC overnight and then heated in an
autoclave at 121.degree. C. for 40 minutes for detoxication).
[0233] (2) In Situ Hybridization
[0234] Mouse (C57BL/6, male, 8-week old) colon was perfusion fixed
using a fixative (from Genostaff), and the colon was then taken and
again fixed with the same fixative, followed by preparing a
paraffin-embedded block. Four-.mu.p slices were prepared from the
resultant paraffin-embedded block, followed by in situ
hybridization using the probes for GT01 and GLP-1. Staining was
performed based on the method of Genostaff Inc. After the
hybridization reaction, the probes were washed before replacement
with TNE (10 mM Tris-HCl (pH 7.6), 500 mM NaCl, 1 mM EDTA) for 10
minutes, followed by decomposing excess probes using 20 mg/ml RNase
(diluted with TNE). Washing was carried out with TNE for 10
minutes, followed by washing with 2.times.SSC, 1.times.SSC, and
0.5.times.SSC at 55.degree. C. for 20 minutes. To detect a signal,
replacement by TBS (100 mM Tris-HCl (pH 7.5), 150 mM NaCl) was
performed for 5 minutes, followed by the reaction of blocking a DIG
antibody at 37.degree. C. for one hour using a 1.5% blocking
reagant (dilution with TBS). Washing was carried out with TBS for 5
minutes, and antibody reaction was performed at room temperature
for one hour using a sheep anti-DIG antibody ((Roche Diagnostics,
Switzerland) 1:500 (dilution with 1.5% blocking reagent)). The
antibody was removed by washing with TBST (100 mM Tris-HCl (pH
7.5), 150 mM NaCl, 0.1% Tween 20), and replacement by NTM (100 mM
Tris-HCl (pH 9.5), 100 mM NaCl, 50 MM MgCl.sub.2) was carried out
for 3 minutes. Then, color development was performed under
microscopic examination using 0.34 mg/ml NBT and 0.18% BCIP
(dilution with NTM), and the color development reaction was
terminated by treatment with a reaction termination solution (10 mM
Tris-HCl, 1 mM EDTA (pH 8.0)) for 10 minutes. After the color
development, the slices were each placed on a slide glass in PBS,
mounted with 90% glycerol (dilution with PBS), and subjected to
microscopic examination using an optical microscope (FIG. 9).
[0235] The human colon sample was also observed using the same
method as that described above (FIG. 10).
[0236] As a result of observation, it was shown that the sites of
expression of GT01 and GLP-1 coincided closely with each other.
EXAMPLE 11
In Vivo Effects of Ligands for GT01 on GLP-1 Release
[0237] Effects of ligands (free fatty acids) for GT01 on GLP-1
release were examined in vivo.
[0238] A 100 nmol/g of fatty acid (.alpha.-linolenic acid, stearic
acid, or octanoic acid) was included in polyethyleneglycol as a
vehicle, and given, through a tube, directly into the stomach of
8-week old male C57/B6 mice (Sankyo Lab) after 24-hour fasting. The
mice were each anaesthetized with diethyl ether 0.5 or 2 hours
after the administration of the fatty acid, followed by sampling
the blood from the portal vein using a heparinized syringe. The
heparizized blood was centrifuged at 4.degree. C. and 1,200.times.g
for 20 minutes to prepare a plasma which was then subjected to
enzyme immunoassay for GLP-1 (Yanaihara Institute Inc.). All
experiments were performed according to an institute-approved
guideline. As a result, the secretion of GLP-1 was induced
particularly when .alpha.-linolenic acid or stearic acid was given
(FIG. 11).
[0239] In addition, the concentration of GLP-1 in the plasma taken
from the portal vein or inferior vena cava was measured using the
same method (FIG. 12).
EXAMPLE 12
An In Vivo Effect of a Ligand for GT01 on Insulin Release
[0240] An effect of a ligand (free fatty acid) for GT01 on insulin
release was examined in vivo.
[0241] A 100 nmol/g of fatty acid (.alpha.-linolenic acid) was
included in polyethyleneglycol as a vehicle, and given, through a
tube, directly into the stomach of 8-week old male C57/B6 mice
(Sankyo Lab) after 24-hour fasting. The mice were each
anaesthetized with diethyl ether 2 hours after the administration
of the fatty acid, followed by sampling the blood from the portal
vein using a heparinized syringe. The heparizized blood was
centrifuged at 4.degree. C. and 1,200.times.g for 20 minutes to
prepare a plasma, in which insulin was quantitated using an enzyme
immunoassay kit of insulin (Morinaga). All experiments were
performed according to an institute-approved guideline. As a
result, the secretion of a higher concentration of insulin was
detected in the plasma sampled from the portal vein than in that
from the inferior vena cava (FIG. 13).
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INDUSTRIAL APPLICABILITY
[0285] The present invention aids in the development of a
pharmaceutical preparation for the lowering of elevated blood sugar
level in diabetes or the like and for the prevention of the
elevation, and particularly also enables the development of a
pharmaceutical preparation suitable for the treatment of patients
whose obese symptoms are marked.
Sequence CWU 1
1
7 1 377 PRT Homo sapiens 1 Met Ser Pro Glu Cys Ala Arg Ala Ala Gly
Asp Ala Pro Leu Arg Ser 1 5 10 15 Leu Glu Gln Ala Asn Arg Thr Arg
Phe Pro Phe Phe Ser Asp Val Lys 20 25 30 Gly Asp His Arg Leu Val
Leu Ala Ala Val Glu Thr Thr Val Leu Val 35 40 45 Leu Ile Phe Ala
Val Ser Leu Leu Gly Asn Val Cys Ala Leu Val Leu 50 55 60 Val Ala
Arg Arg Arg Arg Arg Gly Ala Thr Ala Cys Leu Val Leu Asn 65 70 75 80
Leu Phe Cys Ala Asp Leu Leu Phe Ile Ser Ala Ile Pro Leu Val Leu 85
90 95 Ala Val Arg Trp Thr Glu Ala Trp Leu Leu Gly Pro Val Ala Cys
His 100 105 110 Leu Leu Phe Tyr Val Met Thr Leu Ser Gly Ser Val Thr
Ile Leu Thr 115 120 125 Leu Ala Ala Val Ser Leu Glu Arg Met Val Cys
Ile Val His Leu Gln 130 135 140 Arg Gly Val Arg Gly Pro Gly Arg Arg
Ala Arg Ala Val Leu Leu Ala 145 150 155 160 Leu Ile Trp Gly Tyr Ser
Ala Val Ala Ala Leu Pro Leu Cys Val Phe 165 170 175 Phe Arg Val Val
Pro Gln Arg Leu Pro Gly Ala Asp Gln Glu Ile Ser 180 185 190 Ile Cys
Thr Leu Ile Trp Pro Thr Ile Pro Gly Glu Ile Ser Trp Asp 195 200 205
Val Ser Phe Val Thr Leu Asn Phe Leu Val Pro Gly Leu Val Ile Val 210
215 220 Ile Ser Tyr Ser Lys Ile Leu Gln Thr Ser Glu His Leu Leu Asp
Ala 225 230 235 240 Arg Ala Val Val Thr His Ser Glu Ile Thr Lys Ala
Ser Arg Lys Arg 245 250 255 Leu Thr Val Ser Leu Ala Tyr Ser Glu Ser
His Gln Ile Arg Val Ser 260 265 270 Gln Gln Asp Phe Arg Leu Phe Arg
Thr Leu Phe Leu Leu Met Val Ser 275 280 285 Phe Phe Ile Met Trp Ser
Pro Ile Ile Ile Thr Ile Leu Leu Ile Leu 290 295 300 Ile Gln Asn Phe
Lys Gln Asp Leu Val Ile Trp Pro Ser Leu Phe Phe 305 310 315 320 Trp
Val Val Ala Phe Thr Phe Ala Asn Ser Ala Leu Asn Pro Ile Leu 325 330
335 Tyr Asn Met Thr Leu Cys Arg Asn Glu Trp Lys Lys Ile Phe Cys Cys
340 345 350 Phe Trp Phe Pro Glu Lys Gly Ala Ile Leu Thr Asp Thr Ser
Val Lys 355 360 365 Arg Asn Asp Leu Ser Ile Ile Ser Gly 370 375 2
361 PRT Mus musculus 2 Met Ser Pro Glu Cys Ala Gln Thr Thr Gly Pro
Gly Pro Ser His Thr 1 5 10 15 Leu Asp Gln Val Asn Arg Thr His Phe
Pro Phe Phe Ser Asp Val Lys 20 25 30 Gly Asp His Arg Leu Val Leu
Ser Val Val Glu Thr Thr Val Leu Gly 35 40 45 Leu Ile Phe Val Val
Ser Leu Leu Gly Asn Val Cys Ala Leu Val Leu 50 55 60 Val Ala Arg
Arg Arg Arg Arg Gly Ala Thr Ala Ser Leu Val Leu Asn 65 70 75 80 Leu
Phe Cys Ala Asp Leu Leu Phe Thr Ser Ala Ile Pro Leu Val Leu 85 90
95 Val Val Arg Trp Thr Glu Ala Trp Leu Leu Gly Pro Val Val Cys His
100 105 110 Leu Leu Phe Tyr Val Met Thr Met Ser Gly Ser Val Thr Ile
Leu Thr 115 120 125 Leu Ala Ala Val Ser Leu Glu Arg Met Val Cys Ile
Val Arg Leu Arg 130 135 140 Arg Gly Leu Ser Gly Pro Gly Arg Arg Thr
Gln Ala Ala Leu Leu Ala 145 150 155 160 Phe Ile Trp Gly Tyr Ser Ala
Leu Ala Ala Leu Pro Leu Cys Ile Leu 165 170 175 Phe Arg Val Val Pro
Gln Arg Leu Pro Gly Gly Asp Gln Glu Ile Pro 180 185 190 Ile Cys Thr
Leu Asp Trp Pro Asn Arg Ile Gly Glu Ile Ser Trp Asp 195 200 205 Val
Phe Phe Val Thr Leu Asn Phe Leu Val Pro Gly Leu Val Ile Val 210 215
220 Ile Ser Tyr Ser Lys Ile Leu Gln Ile Thr Lys Ala Ser Arg Lys Arg
225 230 235 240 Leu Thr Leu Ser Leu Ala Tyr Ser Glu Ser His Gln Ile
Arg Val Ser 245 250 255 Gln Gln Asp Tyr Arg Leu Phe Arg Thr Leu Phe
Leu Leu Met Val Ser 260 265 270 Phe Phe Ile Met Trp Ser Pro Ile Ile
Ile Thr Ile Leu Leu Ile Leu 275 280 285 Ile Gln Asn Phe Arg Gln Asp
Leu Val Ile Trp Pro Ser Leu Phe Phe 290 295 300 Trp Val Val Ala Phe
Thr Phe Ala Asn Ser Ala Leu Asn Pro Ile Leu 305 310 315 320 Tyr Asn
Met Ser Leu Phe Arg Asn Glu Trp Arg Lys Ile Phe Cys Cys 325 330 335
Phe Phe Phe Pro Glu Lys Gly Ala Ile Phe Thr Asp Thr Ser Val Arg 340
345 350 Arg Asn Asp Leu Ser Val Ile Ser Ser 355 360 3 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 3 atgtcccctg aatgcgcgcg gg 22 4 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 4 gccagaaata
atcgacaagt ca 22 5 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 5 cgcacccgct ttcccttctt ctc 23 6
25 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 6 agctctttcc ttgatgcctt tgtga 25 7 1389 DNA Mus
musculus 7 cagatgagcg ctctctcaga cagcggcggg cggccgggcg cccggcatgt
cccctgagtg 60 tgcacagacg acgggccctg gcccctcgca caccctggac
caagtcaatc gcacccactt 120 ccctttcttc tcggatgtca agggcgacca
ccggttggtg ttgagcgtcg tggagaccac 180 cgttctgggg ctcatctttg
tcgtctcact gctgggcaac gtgtgtgctc tagtgctggt 240 ggcgcgccgt
cggcgccgtg gggcgacagc cagcctggtg ctcaacctct tctgcgcgga 300
tttgctcttc accagcgcca tccctctagt gctcgtcgtg cgctggactg aggcctggct
360 gttggggccc gtcgtctgcc acctgctctt ctacgtgatg acaatgagcg
gcagcgtcac 420 gatcctcaca ctggccgcgg tcagcctgga gcgcatggtg
tgcatcgtgc gcctccggcg 480 cggcttgagc ggcccggggc ggcggactca
ggcggcactg ctggctttca tatggggtta 540 ctcggcgctc gccgcgctgc
ccctctgcat cttgttccgc gtggtcccgc agcgccttcc 600 cggcggggac
caggaaattc cgatttgcac attggattgg cccaaccgca taggagaaat 660
ctcatgggat gtgttttttg tgactttgaa cttcctggtg ccgggactgg tcattgtgat
720 cagttactcc aaaattttac agatcacgaa agcatcgcgg aagaggctta
cgctgagctt 780 ggcatactct gagagccacc agatccgagt gtcccaacaa
gactaccgac tcttccgcac 840 gctcttcctg ctcatggttt ccttcttcat
catgtggagt cccatcatca tcaccatcct 900 cctcatcttg atccaaaact
tccggcagga cctggtcatc tggccatccc ttttcttctg 960 ggtggtggcc
ttcacgtttg ccaactctgc cctaaacccc atactgtaca acatgtcgct 1020
gttcaggaac gaatggagga agattttttg ctgcttcttt tttccagaga agggagccat
1080 ttttacagat acgtctgtca ggcgaaatga cttgtctgtt atttccagct
aactagcctc 1140 tggtgccagg tgaaccacgg tgtgcatgta aagggagtta
acttcaagga aagcccacca 1200 gtgcgccctg ctttaaaaat acccgacttc
caacagcagg catctacgga gccagcaaat 1260 taaggaatga tcgctcagta
taaaaatatt tttccttaaa agaactttct atgggttcct 1320 tttgtgaact
ttttttagtg tgtttgtaat atgatctagt taataaattt ttatttataa 1380
ctgttccta 1389
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